Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Genetic and morphological identification of filarial worm from Iberian hare in Portugal.

The Iberian hare (Lepus granatensis) is an endemic species of the Iberian Peninsula and the only hare species found in Portugal, although also being present in some areas of Spain. The reduction of wild hare populations due to several ecological and sanitary factors, has been raising growing concerns in the recent years. Despite different helminth species were already described in Iberian hares in Portugal, to this date, no filarial worms have been identified in this species. Furthermore, only a few studies on lagomorphs' onchocercid worms are available, referring to other hosts species of hares and/or rabbits. In this study, we describe the presence of filarial worms in the blood vessels of two adult Iberian hares collected in 2019 in continental Portugal. Morphology and sequencing data from the 12S rRNA, coxI, 18S rRNA, myoHC, hsp70 and rbp1 genes, showed that the filaroid species were genetically related with Micipsella numidica. However, the extension of the genetic differences found with M. numidica suggests that the filaroids specimens under study belong to a new species, that we provisionally named Micipsella iberica n. sp.. The body location of this putative new parasite species and its physiological implications indicate that it may constitute a potential menace to the already fragile Iberian hare justifying, therefore, further investigation regarding the morphological characterization, prevalence and real clinical impact of this new parasite in hares.

Isolation and molecular characterization of Toxoplasma gondii isolated from pigeons and stray cats in Lisbon, Portugal.

Cats and pigeons are important factors in the epidemiology of Toxoplasma gondii as felids are the only definitive hosts that can excrete environmentally resistant oocysts, and pigeons share the same places of cats and humans constituting a good model and indicator of the ground field contamination. We aimed to study the virulence and genotypes of T. gondii isolated from pigeons and stray cats in Lisbon, Portugal. Fresh samples of brain from 41 pigeons and 164 cats revealing antibodies to T. gondii were inoculated in mice. Three isolates (one isolated from a cat and two isolated from pigeons) were virulent in the mouse model. Sag2-based genotyping of T. gondii was achieved in 70.7% (29/41) of samples isolated from pigeons (26 samples were type II, two were type III, and one strain was type I). From the cat brain samples, 50% (82/164) yielded Sag2 positive results, where 72 belonged to genotype II and 10 were no type III (it was not possible to discriminate between type I and II). Further genotyping was obtained by multiplex PCR of 5 microsatellites (TUB2, TgM-A, W35, B17, B18), allowing the identification of two recombinant strains that had been previously identified as type II by Sag2 amplification (one isolated from cat brain and the other from pigeon brain). This is the first evidence of recombinant strains circulating in Portugal and the first report of T. gondii genotyping from cats in this country. This study also highlights the importance of environmental contamination in the synanthropic cycle constituting a potential source of human infection.

Parasite communities in stray cat populations from Lisbon, Portugal.

Stray cats live in high-density colonies in urban areas and pose a health hazard to household cats and humans. In Portugal, information on the parasitic fauna of stray cats is limited and relies mostly on results from faecal analysis. The present survey aimed to determine the prevalence, diversity and intensity of parasites in stray cats from the urban area of Lisbon by means of parasitological necropsy. Internal organs were collected from 162 cats captured in different areas of the city and systematically subjected to parasitological dissection. Helminths were identified by macro- and microscopic examination and protozoa by faecal floatation and sedimentation techniques. The overall prevalence of parasites was 90.7% (95% confidence interval (CI): 85.3-94.6%). A total of 12 parasite species was recorded: Cystoisospora felis (14.2%), Cystoisospora rivolta (46.3%), Sarcocystis sp. (1.2%), Ancylostoma tubaeforme (19.1%), Toxocara cati (38.3%), Ollulanus tricuspis (30.9%), Aelurostrongylus abstrusus (12.4%), Eucoleus aerophilus (0.6%), Taenia taeniaeformis (3.1%), Dipylidium caninum (53.1%), Joyeuxiella pasqualei (15.4%) and Diplopylidium nölleri (3.7%). Overall mean species richness was 2.36 ±  1.52. Helminth mean intensity was highest for O. tricuspis (285.8), followed by D. caninum (42.4), J. pasqualei (14.4), A. tubaeforme (8.1) and T. cati (5.9). The prevalence and variety of parasites found in our sampling are substantially higher than the numbers previously reported in Portugal. Some of the parasites, including T. cati and A. tubaeforme, are zoonotic, which emphasizes the need for parasite control strategies based on demographic containment of stray cat populations in urban areas to promote public health protection.

Isolation of Besnoitia besnoiti from infected cattle in Portugal.

Besnoitia besnoiti, an obligate intracellular protozoan parasite belonging to the phylum apicomplexa, is the causative agent of bovine besnoitiosis. Besnoitiosis is responsible for significant losses in the cattle industry of Africa and Mediterranean countries due to the high morbidity rate, abortion and infertility in males. The acute stage of disease is associated with the proliferative forms (tachyzoites) and is characterized by fever, whimpery, general weakness and swelling of the superficial lymph nodes. During the following chronic stage, a huge number of cysts are formed mainly in the subcutaneous tissues. This process is non-reversible, and chronic besnoitiosis is characterized by hyper-sclerodermia, hyperkeratosis, alopecia and, in bulls, atrophy, sclerosis and focal necrosis that cause irreversible lesions in the testis. In this paper we report on the identification of large cysts in the skin of a cow and a bull in Portugal, which presented loss of hair and enlargement and pachydermis all over the body. The observation of a two-layered cyst wall within the host cell, the encapsulation of the host cell by a large outer cyst wall, and the subcutaneous localization of the cysts within the host, were characteristic for B. besnoiti. The parasites were isolated from the infected animals and successfully propagated in Vero cells without prior passages in laboratory animals. Morphological characterization of B. besnoiti tachyzoites and the amplification of the 149 bp segment from the internal transcribed spacer 1 (ITS1), aided with specific primers, confirmed the identification of B. besnoiti.