Parasitologia

Biological control of undesirable organisms and pathogens often involves the introduction of alien species into new regions. However, alien species themselves pose a potential threat to local ecosystems and economies. The Eastern mosquitofish Gambusia holbrooki is recognised as a dangerous invasive species, but despite this, it is still used for biological control of mosquito larvae, a potential vector of malaria plasmodium transmission to humans, on the Black Sea coast of the Caucasus. We focused on the species composition of helminths in this fish species. We detected adult nematodes Pseudocapillaria (Pseudocapillaria) tomentosa and cestodes Schyzocotyle acheilognathi (formely Bothriocephalus acheilognathi). The above nematode was observed for the first time in fish of the genus Gambusia. Importantly, the cestode S. acheilognathi, which we found in G. holbrooki, is the most successful invasive alien parasite species in freshwaters of the planet and is extremely hazardous to natural ecosystems and aquaculture as it can cause mass mortality of young fish. Thus, the current practice of transferring mosquitofish between water bodies on the Black Sea coast of the Caucasus to control mosquito larvae may contribute to the undesirable spread of a harmful quarantine fish parasite. Full article

Soil-transmitted helminths (STHs), including Ascaris lumbricoides, hookworm species, and Trichuris trichiura, cause significant morbidity worldwide. For an effective proper control of their morbidity, accurate diagnosis method is needed. To this end, a polymerase chain reaction (PCR) method has been developed, but its use remains limited due to the high cost of its implementation, the resources required, and the lack of qualified technicians. The objective of the present analysis is to assess the agreement between the Kato–Katz microscopy method and quantitative real-time PCR (RT-PCR) in the diagnostic of STHs before and after treatment, to decipher the usefulness of either technique for evaluation of the treatment. Methods: Stool samples were collected before and after three- or six-weeks post-treatment from study participants and analyzed using Kato–Katz and RT-PCR methods for the diagnosis of STHs infections. The cure rate (CR) was estimated according to each diagnostic method. Agreement between CRs was tested using the Kappa statistical test. Results: Agreement between Kato–Katz and RT-PCR methods varied regarding the STH species targeted and was different after treatment compared with before treatment. At baseline, the two diagnostic methods showed a moderate agreement (0.45 < K < 0.5) for all STH species, whereas after treatment, concordance decreased slightly (0.11 ≤ K ≤ 0.14) for A. lumbricoides, remained moderate (0.5 ≤ K ≤ 0.53) for T. trichiura, and went from moderate to absent for hookworms. Conclusion: Our findings showed basically a moderate agreement between the Kato–Katz method and RT-PCR. There is a likely association with a moderate proportion of microscopy-positive cases. Reciprocally, a decrease in agreement after treatment was observed with low microscopy-positive cases after treatment, whereby RT-PCR was more likely to detect positive cases than microscopy. Therefore, the agreement is positively associated with an increasing in the number of samples testing positive. Full article

Vector-borne infections persist as a significant issue in both human and animal health. Many of the most common vector-borne infections in the USA, especially tick-borne infections, are known to be zoonotic, including Lyme disease, anaplasmosis, and ehrlichiosis, and these infections may also negatively impact the health of infected animals. Convenient patient-side assays for the detection of antibodies to Borrelia burgdorferi, Anaplasma spp., and Ehrlichia spp., and antigen of Dirofilaria immitis have allowed for the generation of robust and large-scale prevalence data in dogs. Data of similar scale and distribution are not available in cats, and most feline prevalence studies have evaluated a small sample size with limited geographic distribution. The objective of this study was to evaluate the prevalence of antibodies to B. burgdorferi, Anaplasma spp., and Ehrlichia spp., and D. immitis antigen among shelter cats across the USA, a population that is presumably at high risk for ectoparasitism and, consequently, exposure to vector-borne infections. In total, 2232 whole blood samples were collected from shelter cats across four regions of the USA—South, Northeast, Midwest, and West—and were evaluated using the Idexx SNAP^® 4Dx^® Test. Ectoparasites were also opportunistically collected from cats during blood collection and morphologically identified. The prevalence of at least one vector-borne infection was 2.60%, and the nationwide prevalence was 1.88% for B. burgdorferi, 0.54% for Anaplasma spp., 0.09% for Ehrlichia spp., and 0.55% for D. immitis. A total of 1120 ectoparasites were collected from 423 cats, including 27 ticks and 1093 fleas. Although the overall prevalence of the pathogens in this survey is relatively low, we observe that there is an increased exposure risk regionally for some agents, with geographic distributions in this study mostly coinciding with established human and canine distributions. Understanding these findings in an assumed non-protected population of cats allows us to extrapolate the risk to pet cats if they are not provided routine veterinary care, including a broad-spectrum parasite prevention program. Full article

Helminth infections pose a significant global health challenge, as existing drugs often lack efficacy and may be contraindicated in some populations. Progress in the development of new drugs is hindered by the lack of innovative models for use in drug research. Metarhabditis blumi nematodes, which are associated with parasitic otitis in cattle, can severely affect the nervous system, leading to death. The treatment and control of this pathology face similar limitations to those for other parasitic diseases. Our study aimed to standardize M. blumi as a model for evaluating the anthelmintic activity of new drugs. Larvae (L3) and adult worms were treated with the reference drugs albendazole (16 µM) and ivermectin (2.5 µM) diluted in an NGM medium for 24 h, and various parameters were evaluated. Motility and mobility were analyzed using a video tracking and analysis program. Morphological and ultrastructural characterizations were performed after chemical fixation using light and scanning electron microscopy (SEM). The results showed that ivermectin was more effective than albendazole in treating M. blumi adults and L3. The SEM images revealed drug-induced ultrastructural changes. Compared to previous studies using the established Caenorhabditis elegans model, M. blumi demonstrated greater resistance to both albendazole and ivermectin. We conclude that M. blumi is a viable model for drug discovery assays and a valuable new experimental model for various biological studies, highlighting that, unlike C. elegans, M. blumi is associated with parasitism. Full article

The quest for new drug candidates targeting neglected parasitic diseases has become increasingly urgent over the past decades. Advancements in formulating and optimizing drug delivery systems begin with basic research, including direct assays to evaluate the activity of molecules against parasitic stages maintained in laboratories; i.e., promastigotes. In the context of leishmaniasis, an endemic disease worldwide, the cultivation of Leishmania parasites can vary significantly across different laboratories. Factors such as culture media composition, pH, supplementation, and temperature can lead to varied drug responses in in vitro activity assays. This study aims to compile the parameters used in Leishmania spp. promastigotes cultivation protocols described in scientific articles published in indexed journals over the past ten years. The data reveal a lack of uniformity among Leishmania culture protocols, suggesting a potential bottleneck in comparing the leishmanicidal potential of in vitro drug candidates reported by different research groups. This condition is crucial to consider, because viability/inhibition assays should begin with fully-grown, healthy promastigote cultures capable of homogeneous division, thereby producing more reproducible results. Full article

Acanthamoeba keratitis (AK) is a rare but serious infection of the cornea, typically associated with contact lens wear. Here, we present a case of AK caused by the Acanthamoeba genotype T4D/T4e in a patient without identifiable risk factors: a 34-year-old woman who initially presented with signs and symptoms suggestive of herpetic keratitis, and who did not respond to conventional treatment. Corneal culture and targeted metagenomic analysis (18S rRNA, 16S-like rRNA) revealed the presence of an Acanthamoeba species closely related to the ‘Nagington’ strain. Despite intensive anti-Acanthamoeba therapy, complications arose necessitating penetrating keratoplasty. In conclusion, this case underscores the importance of considering Acanthamoeba as a causal agent of keratitis in non-contact-lens wearers. The identification of Acanthamoeba genotype T4D/T4e challenges the previous understanding of its pathogenic potential. Furthermore, it emphasizes the need for ongoing research into the pathogenicity of different Acanthamoeba subtypes. Early diagnosis and treatment are essential for preventing vision-threatening complications associated with AK. Full article

Rhinonyssids are obligate hematophagous mites that parasitize the nasal cavity of domestic and wilds birds worldwide. For the first time, two species of nasal mites of the genus Rhinonyssus from Estonia are described. One species of nasal mite, Rhinonyssus pluvialis Fain et Johnston, 1966, is described and illustrated based on material from Pluvialis apricaria Linnaeus, 1758 (Charadriiformes, Charadriidae). Another species of nasal mite, Rhinonyssus tringae Fain, 1963, is described and illustrated based on material from Tringa glareola Linnaeus, 1758 (Charadriiformes, Scolopacidae). The bird hosts were collected in Estonia, Puhato järv. Full article

Dirofilaria immitis is a mosquito-borne nematode of dogs, other carnivores and, occasionally, humans. Globally, D. immitis infection (which causes heartworm) is typically more prevalent in tropical than temperate regions. In this study, the seroprevalence of D. immitis was determined from a sample of 335 non-stray dogs from four municipalities, two each from the states of Puebla and Guerrero in Mexico, using polyclonal antibodies to detect serum antigens using an enzyme-linked immunosorbent assay (ELISA). The accuracy of the assay was compared with the modified Knott’s test. The polyclonal antibody used in the direct ELISA had a high sensitivity (100%) with variable specificity (98.2–98.8%) in the municipalities of Puebla and Guerrero. The area under the curve for the four municipalities was 1.0, indicating a high accuracy test, with a cut-off value ranging from 0.45 to 0.50. The overall prevalence of D. immitis infection was 17.56% (59 out of 335). The highest prevalence was in Acapulco (24.78%), followed by Chilpancingo (20.93%), Tecamachalco (10.81%) and Quecholac (8.06%). The highest percentage of positive samples was detected in tropical regions (23.12%) and the lowest in temperate regions (9.56%). This study demonstrates that polyclonal anti-D. immitis antibodies can successfully diagnose heartworm-infected dogs and be used to monitor prevalence effectively and develop prevention strategies against Dirofilaria infection. Full article

Hedgehogs, Erinaceus spp., are commonly admitted to rescue centres in European countries. However, there are still few studies on parasitological fauna and treatment possibilities, including for E. roumanicus. This study aimed to evaluate parasitism in 34 hedgehogs directly after their admission to the Budapest Zoo and Botanical Garden Wildlife Rescue Centre, as well as the efficacy of fenbendazole treatment. The Mini-Flotac method was used to quantitatively evaluate and assess the efficacy of treatment with fenbendazole (100 mg/kg PO. BID for 7 days) in five selected individuals. Faecal samples were analysed at D0 and D14 after the beginning of the treatment. Globally, the prevalence of positive animals was 76%. Capillaria spp. were the most prevalent (68%), while coccidia showed a prevalence of 32%. Considering the range of action of this benzimidazole, the treatment provided an efficacy of 100%, eliminating all forms of Capillaria spp. Considering the high number of hedgehogs admitted to rescue centres and the potential effects of parasitism in morbidity and mortality during recovery, it is essential to improve our knowledge with regard to the parasitological fauna of these species and to attain effective treatment protocols. Full article

Cerebral coenurosis affects sheep and goats worldwide and has zoonotic implications. Information on the financial burden and understanding of the disease and control practices may inform control strategies. A cross-sectional study was conducted in Babati and Ngorongoro Districts, Tanzania, to determine economic losses, farmers’ knowledge, and attitudes and practices on the control of coenurosis in small ruminants. A structured questionnaire was administered to 558 households for data collection. The total annual loss was TZS 122.8 M (USD 52.9 K) for Babati and TZS 656.5 M (USD 282.9 K) for Ngorongoro District. The average annual household financial loss was TZS 356.8 K (USD 153.83) for Babati and TZS 3.1 M (USD 1.3 K) for Ngorongoro District. Almost 90% of respondents were aware of coenurosis while less than 1% of the respondents had no knowledge of it at all. The average knowledge score was 5.85 out of 16 (36.6%), which implies a low level of knowledge on coenurosis. About 94% of respondents showed a willingness to participate and pay the control costs. Given the economic loss and favorable attitudes toward control participation, delivery of an educational package on epidemiology and control of coenurosis to livestock keepers may trigger collaborative and sustainable intervention. Full article

The prevalence of endoparasites associated with chaetognath abundance in the coastal waters of the Mexican Central Pacific was studied fortnightly from November 2010 to December 2011. A total of 35 (0.21%) out of 16,407 chaetognaths were found to be parasitized. Five out of twelve chaetognath species (Flaccisagitta enflata, F. hexaptera, Parasagitta euneritica, Serratosagitta pacifica, Zonosagitta bedoti) were found to be parasitized by nine endoparasitic taxa: Protists (two morphotypes), digenean metacercariae [Didymozoidae, Hemiuridae, Parahemiurus sp., Lepocreadiidae, Prosorhynchus sp. (Bucephalidae)], and cestodes (metacestodes) [Tetraphyllidea (two morphotypes)]. Parasagitta. euneritica and Z. bedoti were the most abundant chaetognath species, and Protist sp. 2 and Tetraphyllidea sp. 1 were the most abundant parasites. The highest prevalence for most of the endoparasite species occurred in June, and the values varied according to three hydroclimatic periods: stratified (S), semi-mixed (SM), and mixed (M). Eight non-infected chaetognath species, two parasitized chaetognaths (F. enflata and S. pacifica), and two parasites (Protist sp. 1 and Tetraphyllidea sp. 2) were associated with warm temperatures (S and SM periods); in contrast, P. euneritica, Z. bedoti, parasitized F. hexaptera, and the parasite Tetraphyllidea sp. 1 showed a strong local preference for cooler temperatures, high productivity, and high biomass conditions (M periods). We discovered the occurrence of the digenean Prosorhynchus sp. (Bucephalidae) parasitizing the chaetognath P. euneritica, and this is the first report of Prosorhynchus parasitizing chaetognaths worldwide. We also confirmed the presence of Lepocrediidae (metacercariae larval stage) infecting F. hexaptera, a parasite that had only been recorded infecting other chaetognaths of the Atlantic Ocean. The parasite diversity affecting the chaetognath populations of the Central Mexican Pacific coast likely differs between the offshore, outer slope areas, and the surveyed coastal system. Full article

The aim of this study was to evaluate the isolated and associated use of the nematophagous fungi Pochonia chlamydosporia (isolate VC4) as an ovicide and Duddingtonia flagrans (isolate AC001) as a predator in the control of Taenia saginata eggs. Viable T. saginata eggs were obtained by dissecting mature proglottids from a specimen. For the experimental assay, four groups were formed in microtubes, as follows: (G1—T. saginata eggs/control); (G2—100 T. saginata eggs + VC4); (G3—100 T. saginata eggs + AC001); (G4—100 T. saginata eggs + VC4 + AC001). All the groups were stored in a B.O.D. incubation chamber at a temperature of 27 °C for 15 days and then the contents of the microtubes were analyzed using an optical microscope with a 40x objective. At the end of the assay the treated groups (G2 to G4) showed ovicidal activity (destruction of eggs) compared to the control group (G1). The highest ovicidal percentage was observed in group G2 (eggs + VC4), with 43.3%. In groups G3 and G4 (combination of fungal isolates), the ovicidal percentages were 25.7% and 25.6%, respectively. The results of this study shed light on a new possibility for the combined use of different species of nematophagous fungi, which could be used in the future for environmental biological control of T. saginata eggs. Full article

Endocytosis is a quintessential feature of eukaryotes, and the emergence of endocytosis played a major role in the origin and evolution of eukaryotes. During the early evolution of eukaryotes, phagocytosis and the digestion of prey (i.e., bacteria) combined with the endocytosis of macromolecules opened a new source of nutrients beyond osmotrophy. Pathogenic and commensal protozoa have retained endocytosis as a major mechanism of nutrient acquisition even though, in theory, nutrients could be obtained from the host through osmotrophy. Nearly all pathogenic protozoa exhibit endocytosis and have lysosomal-like compartments that function as digestive vacuoles, and endocytosis appears to play a major role in the acquisition of nutrients. Cryptosporidium is a possible exception that may not exhibit endocytosis. Phagotrophy, however, is only observed in parasites of the intestinal lumen and appears to have been lost in blood and tissue parasites. Overall, the basic features of endocytosis and lysosomes are similar to other eukaryotes. Nonetheless, adaptation to the host has generated some novel features that are specific to certain protozoan lineages. Full article

Background: Neospora caninum is a protozoan parasite and a main cause of abortions in cattle worldwide. However, its role in abortions and decreased fertility in sheep is not completely understood, especially due to the complex, multifactorial etiology of abortions. This study aimed to perform a longitudinal field study to investigate the epidemiology of neosporosis and its effect on fertility in endemic sheep herds. Methods: Serological (IFAT) and clinical (outcome of pregnancy) data from 153 ewe-lambs was collected in four intensive management farms in Israel during three consecutive pregnancies. Results: The seroprevalence in ewe-lambs at different farms varied between 24% and 93%. The overall seroprevalence increased from 50% in ewe-lambs to 96.6% at the end of the third pregnancy. Horizontal infection was observed in all farms, with seroconversion in 59% of seronegative sheep. Abortion rates were lower (p = 0.004) in seropositive ewes in the first pregnancy and not significantly higher in seropositive sheep in consecutive pregnancies. Seropositivity or seroconversion were not associated with abortions or repeated abortions; however, many aborting ewes were removed from the flock. Conclusions: No direct short- or long-term association was found between Neopsora infection and abortions. The variations between flocks and pregnancies suggest a more complex etiology. Full article

Balamuthia mandrillaris is a free-living amoeba (FLA) that causes granulomatous amebic encephalitis (GAE) and skin lesions. Transcriptomic analysis is a powerful tool used to study B. mandrillaris pathogenic infections. However, preliminary tests of RNA extraction showed poor results, so it has become essential to standardize a protocol for high-quality RNA. The present study evaluated 11 RNA extraction protocols based on three commercial kits by making modifications to the temperature and centrifugation times, and by combining kits. Four protocols, namely Q3 (based on QIAGEN RNeasy Mini Kit, with modifications in temperature and centrifugation times), T1 (Invitrogen TRIzol Reagent), T2 (combination of TRIzol and QIAGEN modified protocols) and T3 (combination of TRIzol and PROMEGA SV Total RNA Isolation protocols), presented RNA with good integrity and purity, except for the T1 protocol, which obtained an A260/230 value below the acceptable threshold. High RNA integrity (RIN) values were obtained with the Q3 (9.8), T2 (9.2), and T3 (8.9) protocols, while the T1 protocol obtained a lower RIN value (7.1). The Q3, T2, and T3 protocols obtained high-quality RNA from B. mandrillaris based on the criteria of integrity, purity, and concentration, where the implemented modifications and combinations raised the quality; thus, their use is recommended to obtain accurate results when performing transcriptomic analysis. Full article

During an epidemiological survey following a mortality event of freshwater mussels in 2018 in the Embarrass River, Wisconsin, USA, we identified a novel microsporidian parasite in the ovaries of mucket (Actinonaias ligamentina), plain pocketbook (Lampsilis cardium), and fatmucket (Lampsilis siliquoidea) (Unionidae). Histopathology showed round-to-oval microsporidian spores in the cytoplasm of oocytes in 60% (3/5) of female mucket, 100% (4/4) of female plain pocketbook, and 50% (1/2) of female fatmucket. Using transmission electron microscopy, we found that mature spores were round-to-oval and measured 4.13 ± 0.64 µm (3.14–5.31) long by 2.88 ± 0.37 µm (2.36–3.68) wide. The spores had a thin electron-dense exospore with a spiky “hairy” coat, a thick electron lucent endospore, diplokaryotic nuclei, a polar vacuole, and 27–28 polar filaments arranged in 1–3 rows. Sequencing of the small subunit rRNA produced a 1356 bp sequence most similar to that of Pseudonosema cristatellae (92%), and phylogenetic analysis grouped it within the freshwater Neopereziida. Genetic, morphological, and ultrastructural characteristics did not closely match those of other Pseudonosema spp., and a new genus and species, Hirsutonosema embarrassi n. gen. n. sp., were designated. Additional studies could evaluate host susceptibility, distribution, seasonality, transmission, and lethal or sub-lethal effects of this parasite on freshwater mussels. Full article

Soil-transmitted helminths (STHs) are a group of parasites that are globally distributed and are the most prevalent neglected disease (NTD) in Latin America and the Caribbean (LAC); their presence is associated with health and development problems. In Argentina, their distribution is heterogenous, and there are highly endemic areas in the north of the country. The World Health Organization (WHO) recommends the mass deworming of children as a first-line strategy for the prevention and control of STHs and recently also encourage the development of more sensitive diagnostic tests. The aim of this study was to determine the prevalence of STHs in Tartagal (Salta, Argentina) after four years of deworming interruption with albendazole and ivermectin. A total of 437 fecal samples were analyzed using standard coprological techniques, a subset of which were selected to molecularly typify protozoan parasites; 257 blood samples were analyzed for the presence of specific antibodies to the STH Strongyloides stercoralis. The most prevalent protozoan species were G. intestinalis (19.6–49.2%) and B. hominis (19.1–38.5%). Molecular characterization allowed us to evidence possible zoonotic or human-to-human transmission pathways for Giardia intestinalis or Blastocystis spp., while serology for S. stercoralis proved to be a useful screening tool for monitoring this parasite after treatment. In general, a decrease in the prevalence of STHs was observed in the area, from 60% to 2.9–20% for hookworms and from 51% to 1–9.3% for S. stercoralis four years after treatment, demonstrating the effectiveness and duration of anthelmintic treatment with these two drugs. Full article

Babesiosis, caused by Babesia ovis, is a major seasonal issue in sheep, particularly in countries like Türkiye with high Rhipicephalus bursa tick populations. Previous studies employing various methods such as microscopy, serology, or molecular techniques have reported different epidemiological data concerning ovine babesiosis. Addressing this knowledge gap, our study employed a combined nested PCR (nPCR)/indirect ELISA (iELISA) approach, analyzing blood samples collected from 414 sheep between April and July 2023 using both techniques. nPCR amplified the 18S ribosomal RNA gene of B. ovis and determined a molecular prevalence of 1.9%. Conversely, serological testing using iELISA targeted the BoSA1 antigen and revealed a significantly higher positivity rate of 59.9% for anti-B. ovis antibodies. The temporary presence of Babesia after recovery reduces nPCR sensitivity, resulting in lower molecular prevalence. However, even if Babesia is not present in the host, anti-B. ovis antibodies remain in the serum for a long time and can be detected serologically. Our study underscores the necessity of concurrently employing molecular and serological methods for an accurate assessment of B. ovis prevalence. It highlights the importance of comprehensive epidemiological approaches for effective disease management in sheep populations. Full article

Background: Accurate malaria diagnosis constitutes a challenging task, necessitating the need for the implementation of targeted and effective diagnostic tools. The purpose of the current study was to evaluate the effectiveness of two different molecular methodologies in terms of sensitivity for the detection of External Quality Assessment (EQA) Plasmodium samples. Methods: A total of 104 lyophilized blood samples from 14 different UK-NEQAS (National External Quality Assessment Site) (2016–2021) and eight WHO-NEQAS distributions (2017–2020) were analyzed. An in-house multiplex PCR protocol, followed by single target real-time PCR protocols for all five Plasmodium species, was implemented. Results: The multiplex PCR had a success rate of 10/16 and 20/28 for P. vivax and P. falciparum species, respectively. On the other hand, the respective results for real-time PCR had a success rate of 13/16 (P. vivax), 28/28 (P. falciparum), 5/8 (P. malariae), 8/10 (P. ovale), and 10/14 (P. knowlesi). Plasmodium falciparum samples displayed the highest sensitivity of detection, 0.02 parasites/μL. Plasmodium vivax samples displayed a 0.1 parasites/μL cutoff value, greater than the respective value for whole blood samples, while P. ovale species displayed a respective cutoff value of 0.05 parasites/μL. Due to the limited number of tested samples, data obtained for P. malariae and P. knowlesi species samples were inconclusive. Conclusions: Real-time PCR comprises a credible molecular methodology in terms of sensitivity assessment and detection of low parasitemia levels of Plasmodium sp. in EQA lyophilized blood samples. Full article