Molecular detection of Toxoplasma gondii and Neospora caninum in seabirds collected along the coast of Santa Catarina, Brazil.

Toxoplasma gondii and Neospora caninum are two closely related protozoans that infect a wide range of animals, including birds. However, the occurrence of N. caninum and T. gondii in seabirds is unknown. Therefore, this study aimed to determine the presence of T. gondii and N. caninum DNA in tissue samples of seabirds. Tissue samples of the pectoral muscles, heart, and brain were collected from 47 birds along the coastline of Santa Catarina State, SC, Brazil. The DNA was extracted from the tissues and screened using nested-PCR (nPCR) targeting internal transcribed spacer 1 (ITS1). T. gondii DNA was detected in tissues from seven seabirds (7/47, 14.8%), kelp gull (Larus dominicanus) (5/21), and Manx shearwater (Puffinus puffinus) (2/8). N. caninum DNA was detected in tissues of nine seabirds (9/47, 19.1%), the kelp gull (L. dominicanus) (4/21), Manx shearwater (P. puffinus) (2/8), neotropic cormorant (Phalacrocorax brasilianus) (1/4), brown booby (Sula leucogaster) (1/5), and white-chinned petrel (Procellaria aequinoctialis) (1/1); however, no co-infection was observed. In conclusion, this study showed the circulation of N. caninum and T. gondii in seabirds along the coastline of Santa Catarina State. Further studies are required to clarify the role of these birds in the epidemiology of neosporosis and toxoplasmosis.

Blood Meal Analysis and Molecular Detection of Leishmania DNA in Wild-Caught Sand Flies in Leishmaniasis Endemic Areas of Turkey and Northern Cyprus.

INTRODUCTION: Phlebotomine sand flies (Diptera: Psychodidae) are known as the vector of diseases such as leishmaniasis, bartonellosis and viral diseases. The aim of this study is to detect the host feeding pattern of sand flies in the endemic areas for leishmaniasis in Turkey (Antalya, Kayseri) and Northern Cyprus (TRNC) as well as the presence of Leishmania DNA in the specimens. METHODS: One-hundred seventy-six blood-fed sand fly specimens were examined for blood meal analysis. A SYBR Green-PCR assay was performed with specific forward primers for each host and a universal reverse primer. Primers of human and goat were used together in multiplex PCR while goat and cow were studied separately. ITS-1 qPCR assay was also performed on both blood-fed and non-blood-fed females to detect Leishmania parasites. RESULTS: Blood sources could be detected in 69 out of 176 blood-fed sand fly specimens. The results of blood meal analysis showed that specimens were fed mostly on cows (22.2%) followed by humans (5.7%), goats (2.8%) and dogs (0.6%). Multiple feeding patterns were also detected as human + cow (3.4%), cow + goat (2.8%) and human + goat (1.7%). Five of the blood-fed specimens were Leishmania spp. positive: P. major s.l. (n = 1), P. tobbi (n = 2) were L. tropica positive from Antalya, P. simici was positive for L. infantum from Kayseri and P. papatasi (n = 1) was positive for L. major from Cyprus. Leishmania infection rates were determined as 3.79%, 1.69% and 2.63% among the blood-fed sand fly specimens in Antalya, Kayseri and TRNC, respectively. CONCLUSION: The SYBR-Green-based multiplex PCR assay is a cost-effective and promising tool for blood meal identification of wild-caught sand flies as well as other blood-sucking arthropods. Feeding patterns of important vector species detected in the present study show the high risk in these endemic areas. As a next step, to identify the blood source in a shorter time and to make the test more sensitive, development of this assay to probe-based and multiplex PCR will be also planned.

Development and evaluation of a molecular based protocol for detection and quantification of Cryptosporidium spp. in wastewater.

Infections caused by protozoan parasites are a major public health concern globally. These infections are commonly diagnosed during water-borne outbreaks, necessitating accurate and highly sensitive detection procedures to assure public health protection. Current molecular techniques are challenged by several factors, such as low parasite concentration, inefficient DNA extraction methods, and inhibitors in environmental samples. This study focused on the development and validation of a molecular protocol for DNA extraction, efficient protozoan (oo)cyst recovery and quantification of protozoan parasites from wastewater using droplet digital polymerase chain reaction (ddPCR). Five DNA extraction methods, including commercial kits, custom phenol-chloroform, and in-house modified methods, were evaluated. The efficiency of each method was assessed via spectrophotometric analysis and ddPCR amplification using specific primers. Lastly, the developed protocol was evaluated for the detection and quantification of Cryptosporidium parvum in wastewater from different regions in South Africa. The conventional phenol-chloroform extraction method yielded the highest DNA concentration of 223 (±0.71) ng/µl and detected the highest number of Cryptosporidium parvum (1807 (±0.30) copies/ddPCR reaction) compared to other methods evaluated in this study. Additionally, the phenol-chloroform method demonstrated high sensitivity in extracting DNA from as few as one cyst/L of Cryptosporidium parvum, corresponding to 5.93 copies/ddPCR reaction. It was also observed that analysis of both the filtered supernatant and pellets after centrifugation improves the recovery efficiency of oocysts from wastewater by 10.5%, resulting in a total recovery of 64.1%. This optimized protocol was successfully applied to measure protozoan concentration in wastewater from different regions in South Africa. The improved DNA extraction and quantification method proposed in this study would be effective in monitoring protozoan concentration in the environment, which will help in instituting mitigation measures to reduce water-borne infections.

Occurrence and subtyping of Blastocystis in coypus (Myocastor coypus) in China.

BACKGROUND: Blastocystis is an anaerobic unicellular protist frequently detected in the gastrointestinal tracts of humans and animals worldwide. However, the prevalence and subtype distribution of Blastocystis in the coypu (Myocastor coypus) population have not been reported so far. The aim of this study was to determine the prevalence, genetic characteristics, and zoonotic potential of Blastocystis isolates detected in coypus in China. RESULTS: A total of 308 fecal samples were collected from coypus in seven regions across China and subsequently examined. Blastocystis was detected in 44 (14.3%) specimens by nested PCR amplification of the small subunit ribosomal rRNA (SSU rRNA) gene. Further DNA sequencing and phylogenetic analyses resulted in the identification of two zoonotic known subtypes, ST4 and ST5, and an unknown subtype. ST4 was the most predominant subtype observed in the samples. ST5 infections were only observed in three coypus. Factors that were associated with prevalence of Blastocystis included age, geographical region and subtype. Interestingly, this is the first report about a potentially novel subtype infecting coypus. CONCLUSIONS: This is the first comprehensive report of Blastocystis in M. coypus across a wide geographic range of China. A moderate degree of genetic divergence was observed. The presence of zoonotic subtypes in farmed M. coypus suggests that these animals have the potential to transmit blastocystosis to both humans and domestic animals. These findings provide a better understanding of the genetic diversity of Blastocystis in rodents and contribute towards the establishment of efficient blastocystosis control strategies in the investigated areas.

Phlebotominae Fauna (Diptera: Psychodidae) and Molecular Detection of Leishmania (Kinetoplastida: Trypanosomatidae) in Urban Caves of Belo Horizonte, Minas Gerais, Brazil.

Sand flies are often collected in urban areas, which has several implications for the risk of transmission of Leishmania Ross, 1903, to humans and other mammals. Given this scenario, we describe the sand fly fauna of caves and their surroundings in Mangabeiras Municipal Park (MMP) and Paredão Serra do Curral Park (PSCP), both located in the urban area of Belo Horizonte, Minas Gerais, Brazil, an endemic focus of visceral and cutaneous leishmaniasis. Collections were conducted monthly from November 2011 to October 2012, using CDC light traps exposed for two consecutive nights in four caves and their surroundings. Nonsystematized collections using Shannon traps and active searches were also performed around the caves. The presence of Leishmania DNA in collected female sand flies was evaluated by ITS1-PCR. A total of 857 sand flies representing fourteen species were collected in MMP, of which Evandromyia edwardsi (Mangabeira, 1941) was the most abundant. Leishmania amazonensis was detected in Brumptomyia nitzulescui (Costa Lima, 1932) and Ev. edwardsi, with the latter also having Leishmania braziliensis, Leishmania infantum, and Leishmania sp. A total of 228 sand flies representing four species were collected in PSCP, of which Sciopemyia microps (Mangabeira, 1942) was the most abundant. No females from PSCP were positive for Leishmania-DNA. Studies aimed at describing sand fly faunas of cave environments and detecting Leishmania are essential to understanding the relationship between these insects and this ecotope and assessing and monitoring areas that may pose risks to the health of visitors and employees.

Safety and Tolerability of Mosquito Bite-Induced Controlled Human Infection with Plasmodium vivax in Malaria-Naive Study Participants-Clinical Profile and Utility of Molecular Diagnostic Methods.

BACKGROUND: Plasmodium vivax controlled human malaria infection (PvCHMI) is an important tool for evaluation of drugs, vaccines, and pathologies associated with this parasite. However, there are few data on safety due to limited numbers of PvCHMIs performed. METHODS: We report clinical and laboratory data, including hematological and biochemical profiles and adverse events (AEs), following mosquito bite-induced PvCHMI in malaria-naive study participants. Malaria diagnosis and treatment initiation was based on microscopic analysis of Giemsa-stained slides. Exploratory molecular assays were used to detect parasites using real-time polymerase chain reaction (PCR). RESULTS: AEs were mild to moderate and no study-related severe AEs were observed in any study participants. The majority of symptoms were transient, resolving within 48 hours. Molecular diagnostic methods detected parasitemia in 100% of study participants before malaria diagnosis using microscopy. Of reported AEs, microscopy detected 67%-100%, quantitative PCR 79%-100%, and quantitative real-time reverse-transcription PCR 96%-100% of study participants prior to appearance of symptoms. Almost all symptoms appeared after initiation of treatment, likely as known consequence of drug treatment. CONCLUSIONS: PvCHMI is safe with the majority of infections being detected prior to appearance of clinical symptoms, which can be further alleviated by using sensitive molecular methods for clinical diagnosis. Clinical Trials Registration. NCT01157897.

Contribution to the knowledge on black flies (Diptera: Simuliidae) as vectors of Leucocytozoon (Haemosporida) parasites in Lithuania.

Black flies (Diptera: Simuliidae) are among the most bothersome blood-sucking dipterans causing severe irritation and distress to poultry, wild birds, animals, and humans globally. These insects are vectors of viruses, bacteria, parasitic protozoans, and nematodes of humans and animals. Parasitic protozoa belonging to Haemosporida (Apicomplexa) are distributed worldwide and black flies are the principal vectors of avian haemosporidian parasites of the genus Leucocytozoon, a common parasite of birds. Based on the detection of parasite DNA in insects, 13 black fly species were reported to be potential vectors of Leucocytozoon in Europe. Information about which species of Simulium can play a role in the transmission of Leucocytozoon parasites is insufficient and needs to be developed. The aim of our study was to determine which black fly species are involved in the transmission of Leucocytozoon parasites in the Eastern Europe. The black fly females were collected in Lithuania using entomological net. They were morphologically identified, dissected to prepare salivary glands preparations, and then screened for the presence of Leucocytozoon parasites using microscopy and PCR-based methods. In all, we collected 437 black fly females belonging to eight species. The DNA of Leucocytozoon (genetic lineage lCOCO18) was detected in one of analysed females identified as Simulium maculatum. All salivary gland preparations were negative for the presence of Leucocytozoon sporozoites. Our results included S. maculatum as a potential vector of Leucocytozoon parasites. Increasing the knowledge on vector ecology, behaviour and improving collection methods may be the key to understand the evolution and diversity of these parasites.

Progress in understanding the phylogeny of the Plasmodium vivax lineage.

There has been some controversy about the evolutionary origin of Plasmodium vivax, particularly whether it is of Asian or African origin. Recently, a new malaria species which closely related to ape P. vivax was found in chimpanzees, in addition, the host switches of P. vivax from ape to human was confirmed. These findings support the African origin of P. vivax. Previous phylogenetic analyses have shown the position of P. vivax within the Asian primate malaria parasite clade. This suggested an Asian origin of P. vivax. Recent analyses using massive gene data, however, positioned P. vivax after the branching of the African Old World monkey parasite P. gonderi, and before the branching of the common ancestor of Asian primate malaria parasites. This position is consistent with an African origin of P. vivax. We here review the history of phylogenetic analyses on P. vivax, validate previous analyses, and finally present a definitive analysis using currently available data that indicate a tree in which P. vivax is positioned at the base of the Asian primate malaria parasite clade, and thus that is consistent with an African origin of P. vivax.

Comparative Evaluation of Re 529-Bp Sequence and B1 Gene in the Detection of Toxoplasma gondii Through PCR in Water Samples of Denizli, Turkey.

PURPOSE: While Toxoplasma gondii (T. gondii) infection is asymptomatic in immunocompetent individuals, it is a life-threatening protozoan in immunocompromised individuals. Its water-borne transmission to humans poses a serious public health concern. Polymerase Chain Reaction (PCR) has a considerable potential for the sensitive and specific detection of T. gondii oocysts in waters. METHODS: Comparative evaluation of RE 529-bp sequence and B1 gene to detect T. gondii tachyzoites and oocysts via PCR in agricultural irrigation water taken from downtown Denizli, Turkey and water samples collected from neighborhood fountains was performed for the first time in Turkish context. RESULTS: Based on real-time PCR targeting the B1 genetic markers and RE 529-bp sequence, T. gondii DNA was identified in 6 (16.7%) out of 48 samples collected from agricultural irrigation water. Besides, our PCR analysis did not establish any presence of T. gondii in drinking water samples. CONCLUSION: T. gondii showed lower sensitivity in B1-based PCR than in PCR targeting RE 529-bp sequence.

Evaluation of real-time qPCR-based methods to detect the DNA of the three protozoan parasites Cryptosporidium parvum, Giardia duodenalis and Toxoplasma gondii in the tissue and hemolymph of blue mussels (M. edulis).

The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii can be transmitted to humans through shellfish consumption. No standardized methods are available for their detection in these foods, and the performance of the applied methods are rarely described in occurrence studies. Through spiking experiments, we characterized different performance criteria (e.g. sensitivity, estimated limit of detection (eLD95METH), parasite DNA recovery rates (DNA-RR)) of real-time qPCR based-methods for the detection of the three protozoa in mussel's tissues and hemolymph. Digestion of mussels tissues by trypsin instead of pepsin and the use of large buffer volumes was the most efficient for processing 50g-sample. Trypsin digestion followed by lipids removal and DNA extraction by thermal shocks and a BOOM-based technique performed poorly (e.g. eLD95METH from 30 to >3000 parasites/g). But trypsin digestion and direct DNA extraction by bead-beating and FastPrep homogenizer achieved higher performance (e.g. eLD95METH: 4-400 parasites/g, DNA-RR: 19-80%). Direct DNA recovery from concentrated hemolymph, by thermal shocks and cell lysis products removal was not efficient to sensitively detect the protozoa (e.g. eLD95METH: 10-1000 parasites/ml, DNA-RR ≤ 24%). The bead-beating DNA extraction based method is a rapid and simple approach to sensitively detect the three protozoa in mussels using tissues, that can be standardized to different food matrices. However, quantification in mussels remains an issue.

Efficacy of artesunate-amodiaquine and artemether-lumefantrine for uncomplicated Plasmodium falciparum malaria in Madagascar, 2018.

BACKGROUND: Since 2005, artemisinin-based combination therapy (ACT) has been recommended to treat uncomplicated falciparum malaria in Madagascar. Artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) are the first- and second-line treatments, respectively. A therapeutic efficacy study was conducted to assess ACT efficacy and molecular markers of anti-malarial resistance. METHODS: Children aged six months to 14 years with uncomplicated falciparum malaria and a parasitaemia of 1000-100,000 parasites/µl determined by microscopy were enrolled from May-September 2018 in a 28-day in vivo trial using the 2009 World Health Organization protocol for monitoring anti-malarial efficacy. Participants from two communes, Ankazomborona (tropical, northwest) and Matanga (equatorial, southeast), were randomly assigned to ASAQ or AL arms at their respective sites. PCR correction was achieved by genotyping seven neutral microsatellites in paired pre- and post-treatment samples. Genotyping assays for molecular markers of resistance in the pfk13, pfcrt and pfmdr1 genes were conducted. RESULTS: Of 344 patients enrolled, 167/172 (97%) receiving ASAQ and 168/172 (98%) receiving AL completed the study. For ASAQ, the day-28 cumulative PCR-uncorrected efficacy was 100% (95% CI 100-100) and 95% (95% CI 91-100) for Ankazomborona and Matanga, respectively; for AL, it was 99% (95% CI 97-100) in Ankazomborona and 83% (95% CI 76-92) in Matanga. The day-28 cumulative PCR-corrected efficacy for ASAQ was 100% (95% CI 100-100) and 98% (95% CI 95-100) for Ankazomborona and Matanga, respectively; for AL, it was 100% (95% CI 99-100) in Ankazomborona and 95% (95% CI 91-100) in Matanga. Of 83 successfully sequenced samples for pfk13, no mutation associated with artemisinin resistance was observed. A majority of successfully sequenced samples for pfmdr1 carried either the NFD or NYD haplotypes corresponding to codons 86, 184 and 1246. Of 82 successfully sequenced samples for pfcrt, all were wild type at codons 72-76. CONCLUSION: PCR-corrected analysis indicated that ASAQ and AL have therapeutic efficacies above the 90% WHO acceptable cut-off. No genetic evidence of resistance to artemisinin was observed, which is consistent with the clinical outcome data. However, the most common pfmdr1 haplotypes were NYD and NFD, previously associated with tolerance to lumefantrine.

Malaria parasite species composition of Plasmodium infections among asymptomatic and symptomatic school-age children in rural and urban areas of Kinshasa, Democratic Republic of Congo.

BACKGROUND: Malaria remains a major public health concern in the Democratic Republic of Congo (DRC), and school-age children are relatively neglected in malaria prevalence surveys and may constitute a significant reservoir of transmission. This study aimed to understand the burden of malaria infections in school-age children in Kinshasa/DRC. METHODS: A total of 634 (427 asymptomatic and 207 symptomatic) blood samples collected from school-age children aged 6 to 14 years were analysed by microscopy, RDT and Nested-PCR. RESULTS: The overall prevalence of Plasmodium spp. by microscopy, RDT and PCR was 33%, 42% and 62% among asymptomatic children and 59%, 64% and 95% in symptomatic children, respectively. The prevalence of Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale spp. by PCR was 58%, 20% and 11% among asymptomatic and 93%, 13% and 16% in symptomatic children, respectively. Among P. ovale spp., P. ovale curtisi, P. ovale wallikeri and mixed P. ovale curtisi + P. ovale wallikeri accounted for 75%, 24% and 1% of infections, respectively. All Plasmodium species infections were significantly more prevalent in the rural area compared to the urban area in asymptomatic infections (p < 0.001). Living in a rural as opposed to an urban area was associated with a five-fold greater risk of asymptomatic malaria parasite carriage (p < 0.001). Amongst asymptomatic malaria parasite carriers, 43% and 16% of children harboured mixed Plasmodium with P. falciparum infections in the rural and the urban areas, respectively, whereas in symptomatic malaria infections, it was 22% and 26%, respectively. Few children carried single infections of P. malariae (2.2%) and P. ovale spp. (1.9%). CONCLUSION: School-age children are at significant risk from both asymptomatic and symptomatic malaria infections. Continuous systematic screening and treatment of school-age children in high-transmission settings is needed.

The sand fly (Diptera: Psychodidae) fauna of the urban area of Lassance, Northeast Minas Gerais, Brazil.

The present study aimed to check the sand flies' fauna on the municipality of Lassance, Minas Gerais, Brazil and detect the presence of Leishmania DNA on the female captured and determine the risk areas of the municipality. Sand flies were collected monthly from May 2018 to April 2019 using automatic light traps for 3 consecutive nights. Eight houses were selected as sample points due its previous reports of tegumentary leishmaniasis and/or canine leishmaniasis. The sand fly's fauna found on the present study it's represented by several medical importance species and the most abundant species found were Lutzomyia longipalpis (77.09%) and Nyssomyia intermedia (10.06%). Leishmania infantum DNA was detected in a pool of Lu. longipalpis resulting on a 2.81% of infection rate. By the frequency of the two most abundant species on this study, we developed a risk area map and it draws attention to sample point 6 due to disparate abundance of sand flies at this site (81.81%). Statistical overview shows Lu. longipalpis as dominant species and, still, Non-Metric Multidimensional Scaling analysis reveal high similarity on fauna's diversity on the study area. Our findings suggest that the diversity of sand flies from the municipality of Lassance may promote the circulation of Leishmania infantum parasites putting in risk the habitants and other mammal's species. Still, our study reinforces the necessity of specific studies focused on breed sites of phlebotomine and its' ecology to expand the knowledge about the behaviour of this group of insects applying directly to leishmaniases' epidemiology.

Differential expression and activity of arginine kinase between the American trypanosomatids Trypanosoma rangeli and Trypanosoma cruzi.

Trypanosoma rangeli is a non-virulent hemoflagellate parasite infecting humans, wild and domestic mammals in Central and Latin America. The share of genotypic, phenotypic, and biological similarities with the virulent, human-infective T. cruzi and T. brucei, allows comparative studies on mechanisms of pathogenesis. In this study, investigation of the T. rangeli Arginine Kinase (TrAK) revealed two highly similar copies of the AK gene in this taxon, and a distinct expression profile and activity between replicative and infective forms. Although TrAK expression seems stable during epimastigotes growth, the enzymatic activity increases during the exponential growth phase and decreases from the stationary phase onwards. No differences were observed in activity or expression levels of TrAK during in vitro differentiation from epimastigotes to infective forms, and no detectable AK expression was observed for blood trypomastigotes. Overexpression of TrAK by T. rangeli showed no effects on the in vitro growth pattern, differentiation to infective forms, or infectivity to mice and triatomines. Although differences in TrAK expression and activity were observed among T. rangeli strains from distinct genetic lineages, our results indicate an up-regulation during parasite replication and putative post-translational myristoylation of this enzyme. We conclude that up-regulation of TrAK activity in epimastigotes appears to improve proliferation fitness, while reduced TrAK expression in blood trypomastigotes may be related to short-term and subpatent parasitemia in mammalian hosts.

CRYPTOSPORIDIUM SPP. IN PET DWARF WINTER WHITE RUSSIAN HAMSTERS (PHODOPUS SUNGORIS SUNGORIS) IN CHINA.

Cryptosporidium spp. have been identified in a wide range of hosts, such as humans and domestic and wild animals, while less information about the prevalence of Cryptosporidium spp. in pet hamsters is documented. A total of 351 dwarf winter white Russian hamsters' fecal specimens were collected from 6 pet markets from the cities of Luzhou and Ziyang in Sichuan province in the southwestern part of China. The prevalence of Cryptosporidium spp. determined with nested-PCR amplification of the partial small-subunit (SSU) rRNA gene was 39.32% (138/351). The highest prevalence of Cryptosporidium spp. was in pet market 5 (79.49%, 62/78), followed by pet market 6 (38.64%, 17/44). The lowest prevalence of Cryptosporidium spp. was observed in pet market 3 (14.89%, 7/47). Statistically significant differences in the prevalence of Cryptosporidium spp. were observed among different pet markets (χ2 = 76.386, df = 5, P < 0.05), and a further post hoc test revealed that only pet market 5 was significantly different from other pet markets. Molecular analysis showed that 4 different Cryptosporidium species or genotypes were identified: Cryptosporidium parvum (n = 127), Cryptosporidium chipmunk genotype III (n = 6), Cryptosporidium andersoni (n = 4), and Cryptosporidium wrairi (n = 1). The identification of Cryptosporidium spp. was further tested with the 60-kDa glycoprotein (GP60) gene, and the positive rate was 29.7% (41/138). This is the first molecular report on Cryptosporidium spp. infection in dwarf winter white Russian hamsters in China. With C. parvum and C. andersoni being identified in both humans and pet hamsters, these findings suggest that pet hamsters may be potential reservoirs of zoonotic Cryptosporidium species and subtypes.

THE INFECTION AND SPECIES IDENTIFICATION OF CANINE BABESIA SPP. IN PARTS OF SHAANXI PROVINCE.

Canine babesiosis is a serious disease among tick-borne haemoprotozoan diseases that threaten dog health. To find out the prevalence of canine babesiosis and its main pathogenic species in Shaanxi Province, the study was centered on the infection of babesiosis in dogs in different regions of the Province. First, a total of 367 blood samples were collected in Shaanxi Province, and 53 Babesia nucleic-acid-positive samples were found by polymerase chain reaction (PCR) identification, with a positive rate of 14.44%, and Babesia gibsoni was found by sequencing analysis. Further analysis showed that the prevalence of canine babesiosis was significantly different in 5 regions. There was no significant difference in infection rates between age groups, with the lowest prevalence in young dogs (10.81%) and the highest in adult dogs (17.29%). The infection rate in male dogs was higher than in female dogs. The morbidity of canine Babesia spp. was significantly different between different seasons, with the highest infection rate in autumn (27.78%) and the lowest in winter (6.10%). In conclusion, the epidemicity of canine Babesia spp. in dogs was mainly affected by region and season, and B. gibsoni was the most common canine Babesia spp. within Shaanxi Province in our study. These results provide basic data for the prevention and control of canine babesiosis in this region.

Evaluation of curcumin and CM11 peptide alone and in combination against amastigote form of Iranian strain of L. major (MRHO/IR75/ER) in vitro.

Curcumin (diferuloylmethane) is the main phytochemical of Curcuma longa Linn, an extract of the rhizome turmeric. For thousands of years, turmeric among other natural products has been used as a dietary spice and as a medicinal plant in Asian countries. The present study reports the leishmanicidal activity of curcumin in different concentrations (10 µM, 20 µM, 40 µM). It is also showing the effect of CM11 peptide (8 µM) alone and in combination with curcumin (10 and 20 µM) as a leishmanicidal drug. The experiments were performed with the amastigote form of Leishmania major (MRHO/IR/75/ER) in vitro and the leishmanicidal activity was analyzed after 12 and 24 h of incubation by Giemsa and DAPI staining. Further investigation was done by using semi-quantitative PCR with new designed common primer pair derived from an 18S rRNA gene belonging to the L. major and mouse, which amplified the above-mentioned gene segments simultaneously with different PCR product size. Our findings showed that curcumin had leishmanicidal activity in a dose and time-dependent manner and its lowest effective dose was at concentrations of 40 µM afetr12 h and 10 µM after 24 h. The IC50 value of curcumin against amastigote forms of L. major was 21.12 µM and 11.77 µM after 12 and 24 h, respectively. Treatment of amastigote form with CM11 (8 µM) alone and in combination with curcumin (10 µM and 20 µM) showed less leishmanicidal activity. Interestingly, CM11 in combination with curcumin (10 µM and 20 µM) had even less leishmanicidal effect compared to curcumin alone in the same concentrations (10 µM and 20 µM). The semi-quantitative PCR analysis confirmed the data achieved by Giemsa and DAPI staining and showed that curcumin reduced the PCR product derived from amastigote form in concentration and time-dependent manner compared to the genome of the host cells.

New contribution to epigenetic studies: Isolation of micronuclei with high purity and DNA integrity in the model ciliated protist, Tetrahymena thermophila.

The ciliated protist Tetrahymena thermophila is a well-known model organism with typical nuclear dimorphism containing a somatic macronucleus (MAC) and a germline micronucleus (MIC). The presence in the same cell compartment of two nuclei with distinctly different structural and functional properties provides an ideal model system to explore mechanisms of genome maintenance. Although methods for the isolation of MIC have been available for many years, cross-contamination and DNA degradation remain unresolved. Here, we describe a reliable and quick method to isolate MIC with high purity and DNA integrity in T. thermophila. Different factors are examined to optimize the MIC purification. The MAC contamination ratio in purified MIC is about 0.19% and DNA integrity of purified MIC is maintained. We also establish a more accurate method to detect the contamination rate of nuclei including microscopic observation and PCR detection. This study will facilitate further epigenetic research in Tetrahymena.

Sporadic isolation of Tetratrichomonas species from the cattle urogenital tract.

Tritrichomonas foetus is a venereal trichomonad parasite which causes reproductive issues in cattle. No other trichomonads are known to be urogenital pathogens in cattle, but there are several reports of Tetratrichomonas and Pentatrichomonas isolates of unclear origin from the cattle urogenital tract (UGT) in the Americas. This study reports the first case of a non-T. foetus cattle urogenital trichomonad isolate in Europe. Molecular analysis of the internal transcribed spacer (ITS) 1-5.8S ribosomal RNA-ITS 2 and 18S ribosomal RNA loci suggest that the isolate is a Tetratrichomonas species from a lineage containing other previously described bull preputial isolates. We identified close sequence similarity between published urogenital and gastrointestinal Tetratrichomonas spp., and this is reviewed alongside further evidence regarding the gastrointestinal origin of non-T. foetus isolates. Routine screening for T. foetus is based on culture and identification by microscopy, and so considering other trichomonad parasites of the bovine UGT is important to avoid misdiagnosis.