The transfection of Cryptosporidium represents a major challenge, and current protocols are based on electroporation of freshly excysted sporozoites using a rather large amount of plasmid DNA which typically has a very poor yield. In this study, we report a fast and simple protocol for transfection of Cryptosporidium parvum that takes advantage of the DNA condensing power of the poly cationic polymer polyethylenimine (PEI) and the gene delivery property of the short cell-penetrating peptide octaarginine. Our novel protocol requires a very low amount of plasmid DNA and does not necessitate special laboratory equipment to be performed. Transfection appears to be more efficient in oocysts just triggered for excystation than the excysted sporozoites. Altogether, the application of octaarginine with PEI allows efficient transfection. To the best of our knowledge, this is the first report on an electroporation-free protocol for transfection of sporozoites of a Cryptosporidium species.

Angiostrongylus vasorum, Crenosoma vulpis and Capillaria aerophila are the most common lungworms of domestic and wild canids. We investigated the short- and long-term lungworm prevalence changes in the Swiss fox population with a focus on A. vasorum. Between 2012 and 2017, lungs and hearts of 533 foxes from north-eastern Switzerland were necropsied and blood samples tested for circulating A. vasorum antigen. Angiostrongylus vasorum prevalence increased steadily from 21.5% in 2012 to 81.8% in 2017. In contrast, C. aerophila and C. vulpis prevalences fluctuated between 41.8 and 74.7%, and 3.6 and 14.9%, respectively. Based on 3955 blood samples collected between 1986 and 2017 from three geographic areas and during four time periods, antigen seropositivity increased from 2.4 to 62.0%. In north-eastern Switzerland, seropositivity was initially low (1.9 and 1.7% in the first two time periods) but increased in the following two decades to 22.2 and 62.0%, respectively. Our findings depict the spectacular expansion of A. vasorum in the past three decades. Regionally, the prevalence in foxes increased 4-fold within 6 years in some regions. This underpins the important role of foxes as reservoir hosts, likely explaining the increasing number of cases of canine angiostrongylosis in Switzerland. Our findings are representative of central Europe and may help anticipating future developments in areas where A. vasorum is present but (still) infrequent.

Clonorchis sinensis (C. sinensis) is one of the most serious food-borne parasites, which can lead to liver fibrosis or cholangiocarcinoma. Effective measures for clonorchiasis prevention are still urgently needed. Bacillus subtilis (B. subtilis) is an effective antigen delivery platform for oral vaccines. Chonorchis sinensis serpin (CsSerpin) was proved to be potential vaccine candidates. In this study, CsSerpin3 was displayed on the surface of B. subtilis spore and recombinant spores were orally administrated to BALB/C mice. CsSerpin3-specific IgA levels in faecal, bile and intestinal mucous increased at 4–8 weeks after the first administration compared with those in control groups. The mucus production and the number of goblet cells in intestinal mucosa elevated in B.s-CotC-CsSerpin3 (CotC, coat protein of B. subtilis spore) spores treated group compared to those in blank control. No significant difference in the activities of glutamic-pyruvic transaminase/ alanine aminotransferase and glutamic oxalacetic transaminase/aspartate aminotransferase were observed between groups. There was no side effect inflammation and observable pathological damage in the liver tissue of mice after administration. Moreover, collagen deposition and Ishak score were statistically reduced in B.s-CotC-CsSerpin3 spores treated mice. In conclusion, B. subtilis spores displaying CsSerpin3 could be investigated further as an oral vaccine against clonorchiasis.

Leishmaniasis is a parasitic disease infecting animals and humans. Two clinical forms (Visceral and cutaneous leishmaniasis) and four species are reported to be present in Turkey. Several studies have investigated canine and human leishmaniasis in Turkey but no study was performed to screen the infection among wild rodents, so far. The present study aims to investigate the role of small wild rodents as reservoir animals for Leishmania spp. in different regions of Turkey. Formalin-preserved tissue samples (spleen, liver, lung) of 712 rodents from 30 provinces were screened for the presence of Leishmania spp. DNA. Before DNA extraction, tissues were dried, rehydrated, and homogenated. Leishmania screening in rodent tissues and species determination was performed with a combination of real-time kDNA and ITS1 polymerase chain reaction protocols. Eight (1.12%) out of 712 animals were found to be positive for Leishmania spp. DNA and species typing revealed five L. infantum, two L. tropica and one L. major among positives. Leishmania major and L. infantum DNA were detected in Apodemus spp. from Zonguldak province located in the Western Black Sea Region, while L. tropica DNA was found in Meriones sp. and Gerbillus dasyurus from Adana and Hatay provinces located in Eastern Mediterranean Region of Turkey. The present study is first to report natural infection of L. infantum, L. major and L. tropica in small wild rodents in Turkey, suggesting their possible roles as reservoirs. Further studies are needed for planning epidemiological studies and also for developing rodent control measures in risky endemic areas to break the transmission cycle.

Wildlife species are often host to a diversity of parasites, but our knowledge of their diversity and ecology is extremely limited, especially for reptiles. Little is known about the host-parasite ecology of the Australian lizard, the King's skink (Egernia kingii). In spring of 2015, we carried out a field-based study of a population of King's skinks on Penguin Island (Western Australia). We documented five species of parasites, including two ectoparasitic mites (an undescribed laelapid mite and Mesolaelaps australiensis), an undescribed coccidia species, and two nematode species (Pharyngodon tiliquae and Capillaria sp.). The laelapid mite was the most abundant parasite, infesting 46.9% of the 113 captured lizards. This mite species increased in prevalence and abundance over the course of the study. Infection patterns of both mites varied with lizard life-stage; sub-adults were more commonly infested with laelapid mites than adults or juveniles, and sub-adults and adults were infested by more laelapid mites than juveniles. By contrast, adults had a higher prevalence of M. australiensis than juveniles or sub-adults. Among the gastrointestinal parasites, P. tiliquae was relatively common among the sampled lizards (35.3%). These results give new important information about reptiles as parasite hosts and what factors influence infection patterns.

The genome of Plasmodium falciparum has one of the most skewed base-pair compositions of any eukaryote, with an AT content of 80–90%. As start and stop codons are AT-rich, the probability of finding upstream open reading frames (uORFs) in messenger RNAs (mRNAs) is high and parasite mRNAs have an average of 11 uORFs in their leader sequences. Similar to other eukaryotes, uORFs repress the translation of the downstream open reading frame (dORF) in P. falciparum, yet the parasite translation machinery is able to bypass these uORFs and reach the dORF to initiate translation. This can happen by leaky scanning and/or reinitiation.

In this report, we assessed leaky scanning and reinitiation by studying the effect of uORFs on the translation of a dORF, in this case, the luciferase reporter gene, and showed that both mechanisms are employed in the asexual blood stages of P. falciparum. Furthermore, in addition to the codon usage of the uORF, translation of the dORF is governed by the Kozak sequence and length of the uORF, and inter-cistronic distance between the uORF and dORF. Based on these features whole-genome data was analysed to uncover classes of genes that might be regulated by uORFs. This study indicates that leaky scanning and reinitiation appear to be widespread in asexual stages of P. falciparum, which may require modifications of existing factors that are involved in translation initiation in addition to novel, parasite-specific proteins.

Trypanosoma cruzi, the etiological agent of Chagas disease, releases factors, including antigens from the trans-sialidase (TS) superfamily, which modulate the host immune responses. Tc13 antigens belong to group IV of TSs and are characterized by C-terminal EPKSA repeats. Here, we studied the effect of the Tc13 antigen from the Tulahuén strain, Tc13Tul, on primary cultures of splenocytes from naïve BALB/c mice. Recombinant Tc13Tul increased the percentage of viable cells and induced B (CD19+) lymphocyte proliferation. Tc13Tul stimulation also induced secretion of non-specific IgM and interferon-γ (IFN-γ). The same effects were induced by Tc13Tul on splenocytes from naïve C3H/HeJ mice. In vivo administration of Tc13Tul to naïve BALB/c mice increased non-specific IgG in sera. In addition, in vitro cultured splenocytes from Tc13Tul-inoculated mice secreted a higher basal level of non-specific IgM than controls and the in vitro Tc13Tul stimulation of these cells showed an enhanced effect on IgM and IFN-γ secretion. Our results indicate that Tc13Tul may participate in the early immunity in T. cruzi infection by favouring immune system evasion through B-cell activation and non-specific Ig secretion. In contrast, as IFN-γ is an important factor involved in T. cruzi resistance, this may be considered a Tc13Tul effect in favour of the host.

In areas endemic for Leishmania infantum, an asymptomatic infection may be an indicator of the extent of transmission. The main goal of this study was to evaluate the applicability of measuring circulating immunological biomarkers as an alternative strategy to characterize and monitor L. infantum asymptomatic infections in combination with serological methods. To this end, 179 children from a region endemic for visceral leishmaniasis (VL), aged 1–10 years old, selected from a cross-sectional study, were identified as asymptomatic (n = 81) or uninfected (n = 98) by qPCR and/or serological tests (ELISA using L. infantum soluble antigen and rK39), and, together with serum samples of children diagnosed with VL (n = 43), were subjected to avidity tests and cytokine levels measurement. Avidity rates (AR) ranging from 41 to 70% were found in 29 children (66%) from the asymptomatic group. On the other hand, high AR (above 70%) were observed in 27 children (64%) from the VL group. Logistic Regression and Classification and Regression Tree (CART) analyses demonstrated that lower AR and IFN-γ production associated with higher IL-17A levels were hallmarks in asymptomatic L. infantum infections. Therefore, this study proposes an association of immunological biomarkers that can be used as a complementary strategy for the characterization and monitoring of asymptomatic VL infections in children living in endemic areas.

Toxoplasmosis is a parasitic zoonotic disease caused by Toxoplasma (T.) gondii. Limited data are available on the occurrence of T. gondii in women especially pregnant women in Pakistan. The present study aimed to determine the occurrence and risk factors associated with T. gondii in pregnant and non-pregnant women in Punjab Province, Pakistan. A cross-sectional study was conducted and 593 samples were collected from pregnant (n = 293) and non-pregnant (n = 300) women of District Headquarter Hospitals of Chiniot, Faisalabad, Jhang and Okara, Pakistan. Data related to demographic parameters and risk factors were collected using a pretested questionnaire on blood sampling day. Serum samples were screened for antibodies (IgG) against T. gondii using ELISA. A univariant and binomial logistic regression was applied to estimate the association between seropositive and explanatory variables considering the 95% confidence interval. P value ⩽0.05 was considered statistically significant for all analysis. Out of 593, 44 (7.42%) women were seropositive for T. gondii IgG antibodies. Occupation, age, sampling location, socioeconomic status, contact with cat, pregnancy status and trimester of pregnancy were significantly associated with seropositivity for T. gondii antibodies. Location and trimester of pregnancy were identified as potential risk factors for T. gondii seropositivity based on binomial logistic regression. Toxoplasma gondii is prevalent in pregnant and non-pregnant women. Therefore, now a necessitated awareness is required to instruct the individuals about these infectious diseases (toxoplasmosis) and their control strategies to maintain the health of human population. Moreover, health awareness among public can help the minimization of T. gondii infection during pregnancy and subsequent risk of congenital toxoplasmosis.

Due to the efforts to control schistosomiasis transmission in tropical countries, a large proportion of individuals from endemic areas present low parasite loads, which hinders diagnosis of intestinal schistosomiasis by the Kato-Katz (KK) method. Therefore, the development of more sensitive diagnostic methods is essential for efficient control measures. The aim was to evaluate the accuracy of a real-time polymerase chain reaction (RT-PCR) to detect Schistosoma mansoni DNA in fecal samples of individuals with low parasite loads. A cross-sectional population-based study was conducted in a rural community (n = 257) in Brazil. POC-CCA® was performed in urine and feces were used for RT-PCR. In addition, fecal exams were completed by 18 KK slides, saline gradient and Helmintex techniques. The combined results of the three parasitological tests detected schistosome eggs in 118 participants (45.9%) and composed the consolidated reference standard (CRS). By RT-PCR, 117 out of 215 tested samples were positive, showing 91.4% sensitivity, 80.2% specificity and good concordance with the CRS (kappa = 0.71). RT-PCR identified 86.9% of the individuals eliminating less than 12 eggs/g of feces, demonstrating much better performance than POC-CCA® (50.8%). Our results showed that RT-PCR is a valuable alternative for the diagnosis of intestinal schistosomiasis in individuals with very low parasite loads.

Our present genetic data of Acanthocephala, especially the mitochondrial (mt) genomes, remains very limited. In the present study, the nearly complete mt genome sequences of Sphaerirostris lanceoides (Petrochenko, 1949) was sequenced and determined for the first time based on specimens collected from the Indian pond heron Ardeola grayii (Sykes) (Ciconiiformes: Ardeidae) in Pakistan. The mt genome of S. lanceoides is 13 478 bp in size and contains 36 genes, including 12 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs) and two ribosomal RNA genes (rRNAs). Moreover, in order to clarify the phylogenetic relationship of the genera Centrorhynchus and Sphaerirostris, and to test the systematic position of S. lanceoides in the Centrorhynchidae, the phylogenetic analyses were performed using Bayesian inference and maximum likelihood methods, based on concatenated nucleotide sequences of 12 PCGs, rRNAs and tRNAs. The phylogenetic results further confirmed the monophyly of the order Polymorphida and the paraphyly of the order Echinorhynchida in the class Palaeacanthocephala. Our results also challenged the validity of the genus Sphaerirostris (Polymorphida: Centrorhynchidae) and showed a sister relationship between S. lanceoides and S. picae (Rudolphi, 1819).