Malaria screening: what is the contribution of molecular biology?

INTRODUCTION: According to the World Health Organization, Microscopy is the gold standard for diagnosing malaria. However, the performance of this examination depends on the experience of the microscopist and the level of parasitemia. Thus, molecular biology detection of malaria could be an alternative technique. AIM: evaluate the contribution of molecular biology in detecting imported malaria. METHODS: This was a descriptive, prospective study, including all students, from the Monastir region, and foreigners, from countries endemic to malaria. The study period was from September 2020 to April 2021. Each subject was screened for malaria by three methods: direct microscopic detection of Plasmodium, detection of plasmodial antigens, and detection of plasmodial DNA by nested PCR. RESULTS: Among the 127 subjects screened, only one had a positive microscopic examination for Plasmodium falciparum. Among the 126 subjects with a negative microscopic examination, twelve students had a positive nested PCR result, i.e. 9.5%. Molecular sequencing allowed the identification of ten isolates of Plasmodium falciparum, one Plasmodium malariae and one Plasmodium ovale. Our study showed that the results of nested PCR agreed with those of microscopy in 90.6% of cases. CONCLUSION: Nested PCR seems more sensitive for the detection of low parasitemias. Hence the importance of including molecular biology as a malaria screening tool to ensure better detection of imported cases.

Feline vector-borne haemopathogens in Türkiye: the first molecular detection of Mycoplasma wenyonii and ongoing Babesia ovis DNA presence in unspecific hosts.

BACKGROUND: Cats are hosts and reservoirs for many haemopathogens such as piroplasms, Rickettsia, hemotropic Mycoplasma, Bartonella, Ehrlichia, and Anaplasma, which are transmitted by various vector arthropods and some of which have a zoonotic concern. Although it is noteworthy that the rate of ownership of companion animals has increased in Türkiye in recent years and that cats account for a large proportion of these animals, there is limited research on the vector-borne infectious agents carried by them. The present study aimed to provide a comprehensive molecular epidemiological data and molecular characterization of feline vector-borne haemopathogens (FVBHs), including piroplasms, anaplasmataceae, rickettsias, haemoplasmas, and Bartonella species in Türkiye. In total, 250 feline blood samples were collected from client-owned cats (n = 203) and shelter cats (n = 47) brought to the Small Animal Hospital of Selcuk University, Veterinary Faculty. RESULTS: Overall, 40 (16%) cats were found to be infected with at least one of the investigated haemopathogens and piroplasm, Mycoplasma spp. and Bartonella spp. prevalence was 1.6%, 11.2%, and 4.8%, respectively. No Anaplasma/Ehrlichia spp. and Rickettsia spp. DNA was detected in the investigated feline samples. Sequence analysis revealed that all four piroplasms belonged to Babesia ovis with a 97.93-99.82% nucleotide sequence identity to 18S rRNA gene sequences from Spain and Türkiye, while some sequenced hemoplasmas were Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm) and Mycoplasma wenyonii, and Bartonella spp. were Bartonella henselae and Bartonella koehlerae species. Co-infections with Mycoplasma spp. and Bartonella spp. were also detected in 4 cats (1.6%) in this study, where single infections were predominant. CONCLUSION: This study provides valuable information on zoonotically important feline vector-borne hemopathogens in Türkiye, some of which have received attention under the One Health perspective, and is the first molecular epidemiological study to demonstrate the presence of Babesia ovis, the causative agent of ovine babesiosis, and Mycoplasma wenyonii DNA, the causative agent of bovine haemotropic mycoplasmosis, in cats. Further studies on the roles of such pathogens detected in unspecific hosts and the host specificity of the vectors that transmit them will contribute to the elucidation of this situation.

Detection and molecular identification of Entamoebaspecies in faecal samples from Duhok province, Kurdistan Region, Iraq.

The study involved the estimation of the prevalence of Entamoeba spp. using microscopy and molecular techniques among symptomatic outpatients during April 2021 to March, 2022. Stool samples were collected from 2592 outpatients with amoebiasis symptoms of both sexes and different ages (≤ l to 60). Also, 107 stool samples were taken randomly from asymptomatic individuals and examined microscopically to detect infection with Entamoeba spp. the positive specimens were used for molecular analysis with positive symptomatic samples targeting the 18S rRNA gene by nested PCR. Microscopically 21.68% (562/2592) were positive, for Entamoeba spp. Males showed highest infection rate than females (67.43% vs 32.56%). Ages from 1-10 years showed the highest rate (54.09%), and urban inhabitant had somewhat a higher rate than rural one (58.54% vs 41.45%) which was statistically non-significant(P>0.05). Among asymptomatic individuals, 57% (61/107) were positive for Entamoeba spp. Nested PCR analysis yielded 73% positive samples for Entamoeba spp. with a fragment size of 897 bp. Three fragment sizes were produced, for E. histolytica, E. dispar and E. moshkovskii which were 439, 174 and 553 bps, respectively. Single infection occurred with, E. histolytica in 46%, of symptomatic and 6% of asymptomatic cases, E. dispar in 38% of asymptomatic and 10% of symptomatic cases, E. moshkovskii, reported at very low rate among both groups.

Genetic diversity of Plasmodium malariae in sub-Saharan Africa: a two-marker genotyping approach for molecular epidemiological studies.

Introduction: Plasmodium malariae is the most common non-falciparum species in sub-Saharan Africa. Despite this, data on its genetic diversity is scarce. Therefore, we aimed to establish a P. malariae genotyping approach based on size polymorphic regions that can be easily applied in molecular epidemiological studies. Methods: Four potential genotyping markers, Pm02, Pm09, P. malariae thrombospondin-related anonymous protein (pmtrap), and P. malariae merozoite surface protein fragment 2 (pmmsp1 F2) were amplified via nested PCR and analysed using automated capillary gel electrophoresis. Results: We observed the highest allelic diversity for pmtrap (MOI = 1.61) and pmmsp1 F2 (He = 0.81). Further applying the two markers pmtrap and pmmsp1 F2 on a different sample set of 21 P. malariae positive individuals followed up over one week, we saw a high consistency in their performance. The results show a large complexity and high dynamics of P. malariae infections in the asymptomatic Gabonese study population. Discussion: We successfully implemented a new genotyping panel for P. malariae consisting of only two markers: pmtrap and pmmsp1 F2. It can be easily applied in other settings to investigate the genotype diversity of P. malariae populations, providing further important data on the molecular epidemiology of this parasite species.

Molecular identification of haemoparasites in animals using blood lysate PCR: a quick and inexpensive alternative to purified whole genomic DNA.

Haemoparasitic diseases constitute a significant constraint to economic livestock farming. Diagnostic techniques that are inexpensive, rapid, reliable, and precise are crucial for the management of diseases. In this context, PCR assays are very valuable yet expensive since the samples must be processed before being included in the PCR reaction. Accordingly, the goal of the current study was to lower the PCR costs without jeopardizing the assay's sensitivity and specificity. For that purpose, the alkaline solution was optimized for low cost and quick DNA extraction (blood lysate), and PCR reagents were modified for optimum reaction. In comparison to purified whole blood genomic DNA, the currently developed and optimized blood lysate method was found to be 95.5% less expensive, as well as being equally sensitive and specific for the molecular detection (PCR) of haemoparasites like Babesia, Theileria, Trypanosoma and rickettsiales in cattle, buffaloes, horses, and dogs. The assay was also demonstrated to be quick, less likely to cross-contaminate, and appropriate for use in laboratories with limited resources. Therefore, the currently developed and optimized blood lysate method could serve as a viable alternative to purified whole blood genomic DNA for molecular detection (PCR) of haemoparasites in animals particularly in resource-limited settings.

Cryptosporidium spp. in captive snakes from 26 provinces in China: Prevalence, molecular characterization, and symptoms.

Snakes are sometimes regarded as pets and are used in traditional Chinese medicine. Cryptosporidium spp. are frequently identified in snakes, representing an important pathogen and causing gastrointestinal diseases. Current data indicate that risk factors for infection and patterns of clinical symptom presentation may differ among Cryptosporidium spp. To better understand the infection status by Cryptosporidium spp., fecal samples were collected from 603 asymptomatic and 147 symptomatic snakes in 26 provinces of China. These samples came from Elaphe guttata, Elaphe obsoleta, Pituophis melanoleucus, Thamnophis sirtalis, Lampropeltis getulus, and Heterodon nasicus. The partial small subunit (SSU) rRNA gene was amplified using nested polymerase chain reaction (PCR) to investigate the infection rate of Cryptosporidium spp., and to assess evolutionary relationships and genetic characterization. A prevalence of 20% was recorded in asymptomatic snakes, with age identified as a significant risk factor. In contrast, 70% of symptomatic snakes were positive for Cryptosporidium spp., with Cryptosporidium serpentis and Cryptosporidium varanii (syn. C. saurophilum). Further analysis revealed a potential association between C. serpentis and regurgitation, and C. varanii and diarrhea, while neither species was linked to flatulence. To our knowledge, this is the first study to report Cryptosporidium spp. and associated clinical signs in symptomatic snakes in China. This study aims to enhance the understanding of Cryptosporidium infections, risk factors, and clinical manifestations in snakes, providing data crucial for the control and prevention of cryptosporidiosis.

Title: Cryptosporidium spp. chez les serpents captifs de 26 provinces de Chine : prévalence, caractérisation moléculaire et symptômes. Abstract: Les serpents sont parfois considérés comme animaux de compagnie et sont utilisés en médecine traditionnelle chinoise. Des Cryptosporidium spp. sont fréquemment identifiés chez les serpents, ont un rôle d'agent pathogène important et provoquent des maladies gastro-intestinales. Les données actuelles indiquent que les facteurs de risque d'infection et les schémas de présentation des symptômes cliniques peuvent varier en fonction des espèces de Cryptosporidium. Pour mieux comprendre l'état d'infection par Cryptosporidium spp., des échantillons fécaux ont été collectés auprès de 603 serpents asymptomatiques et 147 serpents symptomatiques dans 26 provinces de Chine. Ces échantillons provenaient d'Elaphe guttata, Elaphe obsoleta, Pituophis melanoleucus, Thamnophis sirtalis, Lampropeltis getulus et Heterodon nasicus. Le gène de l'ARNr de la petite sous-unité partielle (SSU) a été amplifié à l'aide d'une réaction en chaîne par polymérase (PCR) imbriquée pour étudier le taux d'infection par Cryptosporidium spp. et évaluer les relations évolutives et la caractérisation génétique. Une prévalence de 20 % a été trouvée chez les serpents asymptomatiques, l'âge étant identifié comme un facteur de risque important. En revanche, 70 % des serpents symptomatiques étaient positifs à Cryptosporidium spp. avec Cryptosporidium serpentis et Cryptosporidium varanii (syn. C. saurophilum). Une analyse plus approfondie a révélé une association potentielle entre C. serpentis et la régurgitation, et C. varanii et la diarrhée, alors qu'aucune des deux espèces n'était liée aux flatulences. À notre connaissance, il s'agit ici de la première étude à signaler la présence de Cryptosporidium spp. et les signes cliniques associés chez des serpents symptomatiques en Chine. Cette étude vise à améliorer la compréhension des infections à Cryptosporidium, des facteurs de risque et des manifestations cliniques chez les serpents, en fournissant des données cruciales pour le contrôle et la prévention de la cryptosporidiose.

Description and molecular characterisation of Babesia ailuropodae n. sp., a new piroplasmid species infecting giant pandas.

BACKGROUND: Babesia spp. are protozoan parasites that infect the red blood cells of domesticated animals, wildlife and humans. A few cases of giant pandas (a flagship species in terms of wildlife conservation) infected with a putative novel Babesia sp. have been reported. However, comprehensive research on the morphological and molecular taxonomic classification of this novel Babesia sp. is still lacking. This study was designed to close this gap and formally describe this new Babesia sp. infecting giant pandas. METHODS: Detailed morphological, molecular and phylogenetic analyses were conducted to characterise this Babesia sp. and to assess its systematic relationships with other Babesia spp. Blood samples from giant pandas infected with Babesia were subjected to microscopic examination. The 18S ribosomal RNA (18S rRNA), cytochrome b (cytb) and mitochondrial genome (mitogenome) of the new Babesia sp. were amplified, sequenced and assembled using DNA purified from blood samples taken from infected giant pandas. Based on the newly generated 18S rRNA, cytb and mitogenome sequences, phylogenetic trees were constructed. RESULTS: Morphologically, the Babesia sp. from giant pandas exhibited various forms, including round to oval ring-shaped morphologies, resembling those found in other small canine Babesia spp. and displaying typical tetrads. Phylogenetic analyses with the 18S rRNA, cytb and mitogenome sequences revealed that the new Babesia sp. forms a monophyletic group, with a close phylogenetic relationship with the Babesia spp. that infect bears (Ursidae), raccoons (Procyonidae) and canids (Canidae). Notably, the mitogenome structure consisted of six ribosomal large subunit-coding genes (LSU1-6) and three protein-coding genes (cytb, cox3 and cox1) arranged linearly. CONCLUSIONS: Based on coupled morphological and genetic analyses, we describe a novel species of the genus Babesia, namely, Babesia ailuropodae n. sp., which infects giant pandas.

Transfection of a Molecular Clone of Naegleria gruberi rDNA into N. gruberi Trophozoites.

All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While little is known about the CERE, a complete genome sequence analysis of three Naegleria species clearly demonstrates that there are no rDNA cistrons in the nuclear genome. Furthermore, a single DNA origin of replication has been mapped in the N. gruberi CERE, supporting the hypothesis that CERE replicates independently of the nuclear genome. This CERE characteristic suggests that it may be possible to use engineered CERE to introduce foreign proteins into Naegleria trophozoites. As the first step in exploring the use of a CERE as a vector in Naegleria, we developed a protocol to transfect N. gruberi with a molecular clone of the N. gruberi CERE cloned into pGEM7zf+ (pGRUB). Following transfection, pGRUB was readily detected in N. gruberi trophozoites for at least seven passages, as well as through encystment and excystment. As a control, trophozoites were transfected with the backbone vector, pGEM7zf+, without the N. gruberi sequences (pGEM). pGEM was not detected after the first passage following transfection into N. gruberi, indicating its inability to replicate in a eukaryotic organism. These studies describe a transfection protocol for Naegleria trophozoites and demonstrate that the bacterial plasmid sequence in pGRUB does not inhibit successful transfection and replication of the transfected CERE clone. Furthermore, this transfection protocol will be critical in understanding the minimal sequence of the CERE that drives its replication in trophozoites, as well as identifying regulatory regions in the non-ribosomal sequence (NRS).

Cross-species transmission of Cryptosporidium in wild rodents from the southern region of Zhejiang Province of China and its possible impact on public health.

Wild rodents serve as reservoirs for Cryptosporidium and are overpopulated globally. However, genetic data regarding Cryptosporidium in these animals from China are limited. Here, we have determined the prevalence and genetic characteristics of Cryptosporidium among 370 wild rodents captured from three distinct locations in the southern region of Zhejiang Province, China. Fresh feces were collected from the rectum of each rodent, and DNA was extracted from them. The rodent species was identified by PCR amplifying the vertebrate cytochrome b gene. Cryptosporidium was detected by PCR amplification and amplicon sequencing the small subunit of ribosomal RNA gene. Positive samples of C. viatorum and C. parvum were further subtyped by analyzing the 60-kDa glycoprotein gene. A positive Cryptosporidium result was found in 7% (26/370) of samples, involving five rodent species: Apodemus agrarius (36), Niviventer niviventer (75), Rattus losea (18), R. norvegicus (155), and R. tanezumi (86). Their respective Cryptosporidium positive rates were 8.3%, 5.3%, 11.1%, 7.1%, and 7.0%. Sequence analysis confirmed the presence of three Cryptosporidium species: C. parvum (4), C. viatorum (1), and C. muris (1), and two genotypes: Cryptosporidium rat genotype IV (16) and C. mortiferum-like (4). Additionally, two subtypes of C. parvum (IIdA15G1 and IIpA19) and one subtype of C. viatorum (XVdA3) were detected. These results demonstrate that various wild rodent species in Zhejiang were concurrently infected with rodent-adapted and zoonotic species/genotypes of Cryptosporidium, indicating that these rodents can play a role in maintaining and dispersing this parasite into the environment and other hosts, including humans.

Title: Transmission interspécifique de Cryptosporidium chez les rongeurs sauvages de la région sud de la province chinoise du Zhejiang et son impact possible sur la santé publique. Abstract: Les rongeurs sauvages servent de réservoirs à Cryptosporidium et ont des grandes populations à l'échelle mondiale. Cependant, les données génétiques concernant Cryptosporidium chez ces animaux en Chine sont limitées. Ici, nous avons déterminé la prévalence et les caractéristiques génétiques de Cryptosporidium parmi 370 rongeurs sauvages capturés dans trois endroits distincts de la région sud de la province du Zhejiang, en Chine. Des excréments frais ont été collectés dans le rectum de chaque rongeur et l'ADN en a été extrait. L'espèce de rongeur a été identifiée par amplification par PCR du gène du cytochrome b des vertébrés. Cryptosporidium a été détecté par amplification PCR et séquençage d'amplicons de la petite sous-unité du gène de l'ARN ribosomal. Les échantillons positifs de C. viatorum et C. parvum ont ensuite été sous-typés en analysant le gène de la glycoprotéine de 60 kDa. Un résultat positif pour Cryptosporidium a été trouvé dans 7 % (26/370) des échantillons, impliquant cinq espèces de rongeurs : Apodemus agrarius (36), Niviventer niviventer (75), Rattus losea (18), R. norvegicus (155) et R. tanezumi (86). Leurs taux respectifs de positivité pour Cryptosporidium étaient de 8,3 %, 5,3 %, 11,1 %, 7,1 % et 7,0 %. L'analyse des séquences a confirmé la présence de trois espèces de Cryptosporidium : C. parvum (4), C. viatorum (1) et C. muris (1), et de deux génotypes : Cryptosporidium génotype IV de rat (16) et C. mortiferum-like (4). De plus, deux sous-types de C. parvum (IIdA15G1 et IIpA19) et un sous-type de C. viatorum (XVdA3) ont été détectés. Ces résultats démontrent que diverses espèces de rongeurs sauvages du Zhejiang sont simultanément infectées par des espèces/génotypes de Cryptosporidium zoonotiques et adaptés aux rongeurs, ce qui indique que ces rongeurs peuvent jouer un rôle dans le maintien et la dispersion de ce parasite dans l'environnement et d'autres hôtes, y compris les humains.

Prorocentrum canariense sp. nov., a case of pseudo-cryptic speciation in the cosmopolitan dinoflagellate P. compressum (Prorocentrales, Dinophyceae).

The planktonic dinoflagellate Prorocentrum compressum is widespread in warm and temperate seas. A strain identified as P. cf. compressum BEA 0681B isolated from the island of Gran Canaria, NE Atlantic Ocean, showed a divergence in rDNA/ITS phylogenies with respect to P. compressum. The Canarian strain was oval, with an average length-to-width ratio of 1.35, smooth thecal surface with less than 150 thecal pores, including oblique pores, sometimes with a bifurcated opening. In contrast, P. compressum was rounder, with a length-to-width ratio < 1.2, with reticulate-foveate ornamentation and 200-300 pores per valve. We propose Prorocentrum canariense sp. nov. These species clustered as the most early-branching lineage in the clade Prorocentrum sensu stricto. Although this clade mainly contains planktonic species, the closer relatives were the benthic species P. tsawwassenense and P. elegans. Interestingly, P. compressum and P. canariense sp. nov. are widely distributed in temperate and warm seas without an apparent morphological adaptation to planktonic life. The formation of two concentric hyaline mucilaginous walls could contribute to this success. We discuss the use of Prorocentrum bidens to solve the nomenclature issue of P. compressum that was described citing a diatom as basionym.

Diversity of Babesia spp. in skunks from selected states in the United States of America.

Babesia species are intraerythrocytic protozoan parasites that infect a variety of hosts. The goal of this study was to evaluate the piroplasm species present in skunks in various states in the United States and determine whether there was any geographic variation. Spleen, whole blood, or blood on filter paper were received from Pennsylvania, Kentucky, North Carolina, South Carolina, Georgia, Missouri, Louisiana, Texas, Kansas, and California, and were tested for Babesia sp. We tested four species of skunks including striped skunk (Mephitis mephitis, n = 72), eastern spotted skunk (Spilogale putorius, n = 28), western spotted skunk (Spilogale gracilis, n = 15), and hog-nosed skunk (Conepatus leuconotus, n = 11). A PCR assay targeting the 18S rRNA region and cox1 region were used to determine if skunks were infected with piroplasms and for phylogenetic analyses. A total of 48.4% (61/126) of skunks tested positive for a Babesia species. Both the 18S and cox1 analysis supported a skunk-specific Babesia microti-like sp. of carnivores as well as a species in the B. microti complex that is phylogenetically unique from both B. microti of humans and the B. microti-like sp. of carnivores. In the 18S analysis, there was a third species of Babesia in hog-nosed skunks in the western piroplasm group. This study shows that at least three species of piroplasms occur in skunk species in the United States and further highlights the importance of phylogenetic analyses and the use of multiple gene targets when studying piroplasms.

Title: Diversité des Babesia spp. chez des mouffettes provenant d'États sélectionnés des États-Unis. Abstract: Les espèces de Babesia sont des protozoaires parasites intraérythrocytaires qui infectent divers hôtes. Le but de cette étude était d'évaluer les espèces de piroplasmes présentes chez les mouffettes dans divers états des États-Unis et de déterminer s'il existait une variation géographique. Des rates, du sang total ou du sang sur papier filtre ont été reçus de Pennsylvanie, du Kentucky, de Caroline du Nord, de Caroline du Sud, de Géorgie, du Missouri, de Louisiane, du Texas, du Kansas et de Californie, et ont été testés pour Babesia sp. Nous avons testé quatre espèces de mouffettes, dont la mouffette rayée (Mephitis mephitis, n = 72), la mouffette tachetée de l'Est (Spilogale putorius, n = 28), la mouffette tachetée de l'Ouest (Spilogale gracilis, n = 15) et la mouffette à nez plat (Conepatus leuconotus, n = 11). Un test PCR ciblant la région de l'ARNr 18S et la région cox1 a été utilisé pour déterminer si les mouffettes étaient infectées par des piroplasmes et pour des analyses phylogénétiques. Au total, 48,4 % (61/126) des mouffettes ont été testées positives pour une espèce de Babesia. Les analyses du 18S et du cox1 ont toutes deux confirmé une espèce de type Babesia microti de carnivores spécifique aux mouffettes ainsi qu'une espèce du complexe B. microti qui est phylogénétiquement unique à la fois par rapport à B. microti de l'homme et à l'espèce des carnivores. Dans l'analyse 18S, il y avait une troisième espèce de Babesia chez les mouffettes à nez plat du groupe des piroplasmes de l'ouest. Cette étude montre qu'au moins trois espèces de piroplasmes sont présentes chez les espèces de mouffettes aux États-Unis et souligne en outre l'importance des analyses phylogénétiques et de l'utilisation de plusieurs cibles génétiques lors de l'étude des piroplasmes.

No evidence of pathogenicity of Dientamoeba fragilis following detection in stools: A case-control study.

Dientamoeba fragilis is a ubiquitous intestinal parasite with detection in the stools that has become increasingly frequent following the advent of PCR as a routine screening tool. However, the pathogenicity of this parasite is still much debated. In order to assess the potentially pathogenic nature of this protozoan, a retrospective case-control study was carried out between January and December 2020 on patients from Toulouse University Hospital, with the aim of evaluating the potential clinical effects and changes in laboratory parameters linked to the presence and load of D. fragilis in stools. After matching age, sex and mode of care (consultation or hospitalisation), no significant difference was observed in the frequency of clinical signs between the 36 patients who tested positive for Dientamoeba fragilis PCR in their stools and the 72 control patients who were PCR negative for this protozoan. The presence of D. fragilis in the faeces was not associated with changes in laboratory parameters. Furthermore, a high digestive load of D. fragilis had no identifiable impact on clinical and laboratory parameters. Only the concomitant presence of Blastocystis sp. in stools was significantly more frequent in the D. fragilis group (uni- and multivariate analysis). Finally, this study showed no significant difference in clinical or laboratory signs between patients carrying Dientamoeba fragilis and the control group, regardless of the intestinal parasite load, suggesting that D. fragilis could be considered a commensal of the digestive tract.

Title: Aucune preuve de la pathogénicité de Dientamoeba fragilis détecté dans les selles : une étude cas-témoins. Abstract: Dientamoeba fragilis est un parasite digestif ubiquitaire dont la détection dans les selles est devenue de plus en plus fréquente avec l'avènement de la PCR comme outil de détection de routine. Cependant, la pathogénicité de ce parasite est encore très discutée. Afin d'évaluer le caractère potentiellement pathogène de ce protozoaire, une étude rétrospective cas-témoins a été réalisée entre janvier et décembre 2020 sur des patients du CHU de Toulouse, dans le but d'évaluer les effets cliniques et biologiques potentiels associés à la présence et à la charge de D. fragilis dans les selles. Après appariement sur l'âge, le sexe et le mode de prise en charge (consultation ou hospitalisation), aucune différence significative n'a été observée dans la fréquence des signes cliniques entre les 36 patients testés positifs pour la PCR de Dientamoeba fragilis dans les selles et les 72 patients témoins avec une PCR négative pour ce protozoaire. La présence de D. fragilis dans les selles n'était pas associée à des modifications des paramètres biologiques. De plus, une charge digestive élevée de D. fragilis n'avait pas d'impact identifiable sur les paramètres cliniques et biologiques. Seule la présence concomitante de Blastocystis sp. dans les selles était significativement plus fréquente dans le groupe D. fragilis (analyse uni- et multivariée). En conclusion, cette étude n'a pas montré de différence significative concernant les signes cliniques ou biologiques entre les patients porteurs de Dientamoeba fragilis et le groupe témoin, quelle que soit la charge parasitaire digestive, indiquant que D. fragilis pourrait être considéré comme un commensal du tube digestif.

Description of Babesia galileei sp. nov. A Piroplasmid species causing severe disease in domestic cats.

BACKGROUND: Babesiosis is a tick-borne infection caused by piroplasmid protozoa and associated with anemia and severe disease in humans, domestic animals and wildlife. Domestic cats are infected by at least six Babesia spp. that cause clinical disease. METHODS: Infection with a piroplasmid species was detected by microscopy of stained blood smears in three sick cats from Israel. Genetic characterization of the piroplasmid was performed by PCR amplification of the 18S rRNA, cytochorme B (CytB) and heat shock protein 70 (HSP70) genes and the internal transcribed spacer (ITS) locus, DNA sequencing and phylogenetic analysis. In addition, Haemaphysalis adleri ticks collected from two cats were analyzed by PCR for piroplasmids. RESULTS: The infected cats presented with anemia and thrombocytopenia (3/3), fever (2/3) and icterus (1/3). Comparison of gene and loci sequences found 99-100% identity between sequences amplified from different cats and ticks. Constructed phylogenetic trees and DNA sequence comparisons demonstrated a previously undescribed Babesia sp. belonging to the Babesia sensu stricto (clade X). The piroplasm forms detected included pear-shaped merozoite and round-to-oval trophozoite stages with average sizes larger than those of Babesia felis, B. leo and B. lengau and smaller than canine Babesia s.s. spp. Four of 11 H. adleri adult ticks analyzed from cat # 3 were PCR positive for Babesia sp. with a DNA sequence identical to that found in the cats. Of these, two ticks were PCR positive in their salivary glands, suggesting that the parasite reached these glands and could possibly be transmitted by H. adleri. CONCLUSIONS: This study describes genetic and morphological findings of a new Babesia sp. which we propose to name Babesia galileei sp. nov. after the Galilee region in northern Israel where two of the infected cats originated from. The salivary gland PCR suggests that this Babesia sp. may be transmitted by H. adleri. However, incriminating this tick sp. as the vector of B. galilee sp. nov. would require further studies.

hsp70 PCR-RFLP as an alternative tool to identify Sauroleishmania species.

Sauroleishmania spp. comprises one of the four Leishmania subgenera, which has been historically considered a non-pathogenic protozoan of reptiles. However, some strains appear to be transiently infective to mammals, and recent findings have detected these parasites in dogs and humans in areas where leishmaniasis is endemic. Herein, the digestion pattern of PCR-RFLP of the 234 bp-hsp70 fragment was evaluated as a simpler and cheaper tool to distinguish the Sauroleishmania species from the other Leishmania subgenera. As a result, the digestion of the 234 bp-hsp70 fragments with HaeIII produced a banding pattern specific to the four Sauroleishmania strains assessed. This technique could contribute to the identification of Leishmania parasites isolated from sandflies, reptiles, or even mammals in fieldworks as an alternative to the use of laborious and expensive methodologies.

Survey on Perkinsus species in two economic mussels (Mytilus coruscus and M. galloprovincialis) along the coast of the East China Sea and the Yellow Sea.

Perkinsus, a parasitic pathogen of marine bivalves, is widely distributed among various mollusks in numerous countries. However, the prevalence and diversity of Perkinsus species in the two economically important mussels, Mytilus coruscus and M. galloprovincialis, in China remain unknown. The presence of the Perkinsus species was identified in the two mussels sampled along the coast of the East China Sea and the Yellow Sea, using both the alternative Ray's fluid thioglycolate medium (ARFTM) and conventional polymerase chain reaction (PCR). The ARFTM test indicated the presence of Perkinsus-like hypnospores in the two mussels. The diameter of the hypnospores in M. coruscus was significantly smaller than that in M. galloprovincialis. The prevalence of Perkinsus in M. galloprovincialis and M. coruscus ranged from 0 to 37.5% and 0 to 25%, respectively. The mean intensity of Perkinsus in M. galloprovincialis and M. coruscus ranged from 0 to 5.14 and 0 to 4.92, respectively. The PCR assay showed that the prevalence of Perkinsus spp. in M. galloprovincialis and M. coruscus was 0 to 25.0% and 0 to 12.5%, respectively. The homology analysis of the newly obtained internal transcribed spacer (ITS) sequences of Perkinsus revealed the highest identity of 100% with P. beihaiensis. The phylogenetic analysis indicated that the Perkinsus isolates from the two mussels were clustered with P. beihaiensis. The results of the molecular biology indicated that only P. beihaiensis was detected in the two mussels. The highest prevalence of P. beihaiensis was observed in Liaoning province (Dalian, 20.83%), followed by Shandong province, Zhejiang province and Fujian province. Consequently, it is recommended that surveillance should be conducted in Dalian, where the prevalence and mean intensity of P. beihaiensis in M. galloprovincialis are the highest.

Molecular characterizations of Cryptosporidium spp. in brown rat (Rattus norvegicus) from an animal feedlot in Xinjiang, China.

Cryptosporidium infection is a common occurrence in rodents worldwide. In this study, 435 wild brown rats were captured from an animal feedlot in Xinjiang, China, with a fecal sample obtained directly from the rectal contents of each rat. The DNA extracted from these fecal samples was analyzed for Cryptosporidium spp. using PCR targeting the SSU rRNA gene. The prevalence of Cryptosporidium infection in brown rats was found to be 5.5% (24 out of 435). Interestingly, the infection rates varied among different animal enclosures, with rates of 0% in the chicken coop (0/51), cowshed (0/3), and varying rates in other areas including the sheepfold (6.1%, 6/98), the pigsty (7.6%, 10/132), the dovecote (7.0%, 5/71), and outdoor environments (3.8%, 3/80). The study identified three species and one genotype of Cryptosporidium, namely C. occultus (n = 10), C. parvum (n = 4), C. ditrichi (n = 1), and Cryptosporidium rat genotype IV (n = 9). Additionally, two of the C. parvum isolates were successfully subtyped as IIdA19G1 (n = 2) at the gp60 gene. These results offer valuable insights into the prevalence and genetic diversity of Cryptosporidium in brown rats within the region.

Genetic characterization of Cryptosporidium spp. in the North African hedgehog (Atelerix algirus) in the Canary Islands, Spain.

The North African hedgehog (Atelerix algirus) is an introduced species from Northwest Africa and is currently distributed in the Canary Islands. This species of hedgehog has been studied as a reservoir of enteropathogens, including Cryptosporidium spp. However, there are no data at species level. Therefore, the aim of the present study was to identify the Cryptosporidium species present in a population of hedgehogs (n = 36) in the Canary Islands. Molecular screening was performed using conventional polymerase chain reaction (PCR) targeting the small subunit ribosomal RNA (18S rRNA) gene of Cryptosporidium spp. Seven of the 36 fecal samples (19.45%) were positive and confirmed by nested PCR targeting the 18S rRNA gene and Sanger sequencing. Cryptosporidium parvum and Cryptosporidium muris were identified in 11.1% (4/36) and 5.6% (2/36) of the samples, respectively, while one sample could only be identified at the genus level. The zoonotic subtypes IIdA15G1 (n = 1), IIdA16G1b (n = 1), and IIdA22G1 (n = 1) of C. parvum were identified by nested PCR followed by analysis of the 60 kDa glycoprotein (gp60) gene sequence. This study is the first genetic characterization of Cryptosporidium spp. in A. algirus, identifying zoonotic species and subtypes of the parasite.

Unveiling Blastocystis epidemiology in Morocco: subtype diversity among clinical patients with and without gastrointestinal manifestations in the Meknes region.

Blastocystis is an intestinal protist frequently identified in humans and other animals, though its clinical significance remains controversial. This study aimed to determine the prevalence and genetic diversity of Blastocystis in faecal samples from symptomatic (n = 55) and asymptomatic (n = 50) individuals seeking medical care in Meknes, Morocco. Detection of the protist was accomplished through coproparasitological examination and culture in Jones medium. Culture-positive samples were subjected to molecular analyses (PCR and Sanger sequencing) based on sequences of the small subunit ribosomal RNA gene. Epidemiological questionnaires on demographics and potential risk factors were collected from participating patients. The overall Blastocystis infection rate was 51.4% (54/105), with no differences between symptomatic (52.7%, 29/55) and asymptomatic (50.0%, 25/50) individuals. Sequence analyses identified three Blastocystis subtypes, with ST3 being the most prevalent (42.0%), followed by ST1 (34.0%), and ST2 (12.0%). Regarding intra-subtype diversity, allele 4 was found within ST1; alleles 11/12 and alleles 34/36 (alone or in combination) were identified within ST2 and ST3 respectively. Allele 34 in ST3 (40.8%) and allele 4 in ST1 (34.7%) were the most common genetic variants circulating in the surveyed clinical population. A statistically significant association between ST2 and the presence of flatulence was observed. This is the first study assessing the epidemiology and genetic diversity of Blastocystis sp. in the Meknes region, Morocco.

Emergence of imported cutaneous leishmaniasis caused by Leishmania major: a case series from Kerala, India.

Cutaneous leishmaniasis (CL) is often considered a 'great imitator' and is the most common form of leishmaniasis. The Leishmania species responsible for CL varies among countries, as these species exhibit specific distribution patterns. The increased mobility of people across countries has resulted in the imported incidences of leishmaniasis caused by non-endemic species of Leishmania. During 2023, we confirmed three CL cases caused by L. major from Kerala, India, and upon detailed investigation, these were identified to be imported from the Middle East and Kazakhstan regions. This is the first report of CL caused by L. major from Kerala. The lesion morphology, detection of anti-rK 39 antibody and Leishmania parasite DNA from the blood samples were the unique observations of these cases. Kerala, being an emerging endemic zone of visceral leishmaniasis (VL) and CL, the imported incidences of leishmaniasis by non-endemic species can pose a significant threat, potentially initiating new transmission cycles of leishmaniasis caused by non-endemic species.

The first study of the prevalence and genetic diversity of Theileria equi and Babesia caballi in horses in Russia.

Equine piroplasmosis (EP) is a global worldwide infection, which can lead to the death of animals. Despite the causative agents of EP being well studied, there are no data on the distribution and genetic characteristics of EP agents in any region of Russia. In this study, blood samples from 750 horses from Novosibirsk province, Irkutsk province, and Altai region of Russian Siberia were examined for the presence of EP agents. Theileria equi and Babesia caballi were detected in all examined regions, with mean prevalence rates of 60.4% and 7.2%, respectively. The identified pathogens were genetically characterized by the 18S rRNA gene. The determined T. equi sequences were highly conserved and belonged to genotypes A and E, with genotype E being found in 88.6% of genotyped samples. In contrast to T. equi, B. caballi sequences were genetically diverse. Seven sequence variants of B. caballi were identified, and only two of them matched known sequences from the GenBank database. The determined B. caballi sequences belonged to four distinct branches within genotype A. Mixed infections with several variants of B. caballi or with T. equi and B. caballi were common. The conducted phylogenetic analysis based on all available B. caballi sequences of the 18S rRNA gene (> 900 bp) from GenBank and from this study first demonstrated the presence of five monophyletic clusters within genotype A and three clusters within genotype B. Thus, the genetic study of B. caballi from Siberia has significantly expanded the data on the genetic diversity of this pathogen.