Local terrestrial snails as natural intermediate hosts of the zoonotic parasite Angiostrongylus cantonensis in the new European endemic area of Valencia, Spain.

AIM: The rat lungworm, Angiostrongylus cantonensis, has recently been found in the city of Valencia, parasitizing rats, Rattus norvegicus and Rattus rattus, its natural definitive hosts. This is the first finding of this zoonotic nematode in continental Europe. After informing local and national health authorities, the collection of local terrestrial snails took place with the aim of elucidating their potential role as intermediate hosts of A. cantonensis. METHODS AND RESULTS: A total of 145 terrestrial snails, belonging to the species Cernuella virgata, Cornu aspersum, Eobania vermiculata, Otala punctata, Pseudotachea splendida, Rumina decollata and Theba pisana, were randomly collected between May and December 2022 in public gardens, parks and orchards in six districts of Valencia, in five of which A. cantonensis had been reported previously in rats. Once collected and identified, the snails were frozen at -20°C. Subsequently, the DNA was isolated and screened by PCR using specific primers targeting the A. cantonensis COI gene. Seven individual snails, belonging to the species C. virgata, C. aspersum and T. pisana, were positive, for an overall prevalence of 4.8%. The PCR product from one of them was sequenced by Sanger sequencing. CONCLUSIONS: The three positive terrestrial snail species are among the edible species that are frequently included in various dishes in Spain. C. virgata is reported as a previously unrecorded intermediate host and should be added to the list of more than 200 species of terrestrial snails that have been reported worldwide as intermediate hosts of the rat lungworm. Considering that these terrestrial snails may release infective larvae of A. cantonensis on leafy green vegetables on which they feed and during their handling and preparation for consumption, prophylactic measures to prevent human neuroangiostrongyliasis in Valencia and other regions to which this zoonotic parasite may spread are recommended.

Peripheral inflammation-induced changes in songbird brain gene expression: 3' mRNA transcriptomic approach.

Species-specific neural inflammation can be induced by profound immune signalling from periphery to brain. Recent advances in transcriptomics offer cost-effective approaches to study this regulation. In a population of captive zebra finch (Taeniopygia guttata), we compare the differential gene expression patterns in lipopolysaccharide (LPS)-triggered peripheral inflammation revealed by RNA-seq and QuantSeq. The RNA-seq approach identified more differentially expressed genes but failed to detect any inflammatory markers. In contrast, QuantSeq results identified specific expression changes in the genes regulating inflammation. Next, we adopted QuantSeq to relate peripheral and brain transcriptomes. We identified subtle changes in the brain gene expression during the peripheral inflammation (e.g. up-regulation in AVD-like and ACOD1 expression) and detected co-structure between the peripheral and brain inflammation. Our results suggest benefits of the 3'end transcriptomics for association studies between peripheral and neural inflammation in genetically heterogeneous models and identify potential targets for the future brain research in birds.

Extracellular vesicles of Trypanosoma cruzi and immune complexes they form with sialylated and non-sialylated IgGs increase small peritoneal macrophage subpopulation and elicit different cytokines profiles.

American trypanosomiasis, or Chagas disease, is caused by the protozoan parasite Trypanosoma cruzi and is characterized by the presence of cardiac or gastrointestinal symptoms in a large number of patients during the chronic phase of the disease. Although the origin of the symptoms is not clear, several mechanisms have been described involving factors related to T. cruzi and the host immune response. In this sense, the extracellular vesicles (EVs) secreted by the parasite and the immune complexes (ICs) formed after their recognition by host IgGs (EVs-IgGs) may play an important role in the immune response during infection. The aim of the present work is to elucidate the modulation of the immune response exerted by EVs and the ICs they form by analyzing the variation in the subpopulations of small and large peritoneal macrophages after intraperitoneal inoculation in mice and to evaluate the role of the sialylation of the host IgGs in this immunomodulation. Both macrophage subpopulations were purified and subjected to cytokine expression analysis by RT-qPCR. The results showed an increase in the small peritoneal macrophage subpopulation after intraperitoneal injection of parasite EVs, but a greater increase in this subpopulation was observed when sialylated and non-sialylated ICs were injected, which was similar to inoculation with the trypomastigote stage of the parasite. The cytokine expression results showed the ability of both subpopulations to express inflammatory and non-inflammatory cytokines. These results suggest the role of free EVs in the acute phase of the disease and the possible role of immune complexes in the immune response in the chronic phase of the disease, when the levels of antibodies against the parasite allow the formation of immune complexes. The differential expression of interleukins showed after the inoculation of immune complexes formed with sialylated and non-sialylated IgGs and the interleukins expression induced by EVs, demonstrates that the IgG glycosilation is involved in the type of immune response that dominates in each of the phases of the Chagas disease.

Update on the First Finding of the Rat Lungworm, Angiostrongylus cantonensis, in Rattus spp. in Continental Europe, Valencia, Spain, 2022.

The rat lungworm, Angiostrongylus cantonensis, is an emerging parasite that can cause eosinophilic meningitis in humans. Over the past 60 years, the worm has greatly expanded its original Asian distribution to most tropical and subtropical areas of the world, mostly by traveling on ships with its definitive hosts, rats. Angiostrongylus cantonensis was recently found for the first time in Continental Europe, specifically in 3 (2 Rattus norvegicus and 1 Rattus rattus) out of 27 rats trapped in the sewer system of the city of Valencia, Spain. Updating the investigation, the parasite has subsequently been found in 8 (5 R. norvegicus and 3 R. rattus) out of 94 rats analyzed. The highest prevalence of infection (20%) was obtained in rats trapped in the orchards that surround the city, where both snails and slugs (intermediate hosts) abound, and where vegetables consumed in Valencia, other parts of Spain, and abroad, are produced. The presence of the parasite in rats does not necessarily mean that the disease it causes becomes a relevant public health concern since it strongly depends on the food habits of the population at risk. If proper precautions are taken, the risk of acquiring neuroangiostrongylosis should be minimal.

Parasite Fauna and Coinfections in Urban Rats Naturally Infected by the Zoonotic Parasite Angiostrongylus cantonensis.

When the zoonotic parasite of rodents that can cause human neuroangiostrongyliasis, i.e., Angiostrongylus cantonensis, is found in its natural definitive hosts, it is usually reported in isolation, as if the rat lungworm were the only component of its parasite community. In this study, we report the coinfections found in rats naturally infected by A. cantonensis in urban populations of Rattus norvegicus and Rattus rattus in Valencia, Spain. In addition to the rat lungworms, which were found in 14 of the 125 rats studied (a prevalence of 11.20%), 18 other parasite species (intestinal and tissular protists, microsporidia and helminths) were found, some of them with high burdens. Fourteen of these nineteen species found are potential zoonotic parasites, namely Blastocystis, Giardia duodenalis, Cryptosporidium spp., Enterocytozoon bieneusi, Encephalitozoon hellem, Toxoplasma gondii, Brachylaima spp., Hydatigera taeniaeformis s.l. larvae, Hymenolepis nana, Hymenolepis diminuta, Angiostrongylus cantonensis, Calodium hepaticum, Gongylonema neoplasticum and Moniliformis moniliformis. The total predominance of coinfected rats as well as their high parasite loads seem to indicate a trend towards parasite tolerance.

Cannabinoid receptor 2 evolutionary gene loss makes parrots more susceptible to neuroinflammation.

In vertebrates, cannabinoids modulate neuroimmune interactions through two cannabinoid receptors (CNRs) conservatively expressed in the brain (CNR1, syn. CB1) and in the periphery (CNR2, syn. CB2). Our comparative genomic analysis indicates several evolutionary losses in the CNR2 gene that is involved in immune regulation. Notably, we show that the CNR2 gene pseudogenized in all parrots (Psittaciformes). This CNR2 gene loss occurred because of chromosomal rearrangements. Our positive selection analysis suggests the absence of any specific molecular adaptations in parrot CNR1 that would compensate for the CNR2 loss in the modulation of the neuroimmune interactions. Using transcriptomic data from the brains of birds with experimentally induced sterile inflammation we highlight possible functional effects of such a CNR2 gene loss. We compare the expression patterns of CNR and neuroinflammatory markers in CNR2-deficient parrots (represented by the budgerigar, Melopsittacus undulatus and five other parrot species) with CNR2-intact passerines (represented by the zebra finch, Taeniopygia guttata). Unlike in passerines, stimulation with lipopolysaccharide resulted in neuroinflammation in the parrots linked with a significant upregulation of expression in proinflammatory cytokines (including interleukin 1 beta (IL1B) and 6 (IL6)) in the brain. Our results indicate the functional importance of the CNR2 gene loss for increased sensitivity to brain inflammation.

Autochthonous Angiostrongylus cantonensis Lungworms in Urban Rats, Valencia, Spain, 2021.

To determine the role of rats as potential reservoirs of zoonotic parasites, we examined rats trapped in urban sewers of Valencia, Spain, in 2021. Morphologic and molecular identification and sequencing identified autochthonous Angiostrongylus cantonensis nematodes, the most common cause of human eosinophilic meningitis, in pulmonary arteries of Rattus norvegicus and R. rattus rats.

Absence of Toxoplasma gondii in 100% Iberian products from experimentally infected pigs cured following a specific traditional process.

Infection with Toxoplasma gondii in humans has usually been related to the consumption of raw, undercooked or cured meat. Our study is based on the detection of T. gondii in cured legs and shoulders made from 100% Iberian sows fed mainly with acorn and raised as outdoor livestock in Aracena (Spain), which having been elaborated following a specific curing process (time period and location). An outdoor farm with a total of 636 animals was studied, showing a seroprevalence of 10% for the parasite T. gondii. Twenty individuals were chosen to be experimentally infected and slaughtered 60 days post-infection. Their legs and shoulders were processed to make 100% Iberian ham legs and shoulders. The meat ready to be eaten was analyzed by quantification and viability assays using magnetic capture real-time qPCR and bioassay techniques proving that this specific traditional "Cinco Jotas" curing process 100% Iberian ham is strong enough to eliminate the parasite T. gondii, resulting in a safe product for consumers.

Leishmaniasis in Norway Rats in Sewers, Barcelona, Spain.

We detected Leishmania infantum in 98 Norway rats (Rattus norvegicus) trapped in parks and sewers of Barcelona, Spain. The 84 rats from the sewers showed a prevalence of 33.3% and up to 2,272 estimated parasites. These results, in the most abundant potential reservoir in cities, is of public health concern.

Nest predation risk modifies nestlings' immune function depending on the level of threat.

Predation risk is thought to modify the physiology of prey mainly through the stress response. However, little is known about its potential effects on the immunity of animals, particularly in young individuals, despite the importance of overcoming wounding and pathogen aggression following a predator attack. We investigated the effect of four progressive levels of nest predation risk on several components of the immune system in common blackbird (Turdus merula) nestlings by presenting them with four different calls during 1 h: non-predator calls, predator calls, parental alarm calls and conspecific distress calls to induce a null, moderate, high and extreme level of risk, respectively. Nest predation risk induced an increase in ovotransferrin, immunoglobulin and the number of lymphocytes and eosinophils. Thus, the perception of a potential predator per se could stimulate the mobilization of a nestling's immune function and enable the organism to rapidly respond to the immune stimuli imposed by a predator attack. Interestingly, only high and extreme levels of risk caused immunological changes, suggesting that different immunological parameters are modulated according to the perceived level of threat. We also found a mediator role of parasites (i.e. Leucocytozoon) and the current health status of the individual, as only nestlings not parasitized or in good body condition were able to modify their immune system. This study highlights a previously unknown link between predation risk and immunity, emphasizing the complex relationship among different selective pressures (predation, parasitism) in developing organisms and accentuating the importance of studying predation from a physiological point of view.

Functionalized immunostimulating complexes with protein A via lipid vinyl sulfones to deliver cancer drugs to trastuzumab-resistant HER2-overexpressing breast cancer cells.

BACKGROUND: Around 20%-30% of breast cancers overexpress the proto-oncogene human epidermal growth receptor 2 (HER2), and they are characterized by being very invasive. Therefore, many current studies are focused on testing new therapies against tumors that overexpress this receptor. In particular, there exists major interest in new strategies to fight breast cancer resistant to trastuzumab (Tmab), a humanized antibody that binds specifically to HER2 interfering with its mitogenic signaling. Our team has previously developed immunostimulating complexes (ISCOMs) as nanocapsules functionalized with lipid vinyl sulfones, which can incorporate protein A and bind to G immunoglobulins that makes them very flexible nanocarriers. METHODS AND RESULTS: The aim of this in vitro study was to synthesize and evaluate a drug delivery system based on protein A-functionalized ISCOMs to target HER2-overexpressing cells. We describe the preparation of ISCOMs, the loading with the drugs doxorubicin and paclitaxel, the binding of ISCOMs to alkyl vinyl sulfone-protein A, the coupling of Tmab, and the evaluation in both HER2-overexpressing breast cancer cells (HCC1954) and non-overexpressing cells (MCF-7) by flow cytometry and fluorescence microscopy. Results show that the uptake is dependent on the level of overexpression of HER2, and the analysis of the cell viability reveals that targeted drugs are selective toward HCC1954, whereas MCF-7 cells remain unaffected. CONCLUSION: Protein A-functionalized ISCOMs are versatile carriers that can be coupled to antibodies that act as targeting agents to deliver drugs. When coupling to Tmab and loading with paclitaxel or doxorubicin, they become efficient vehicles for the selective delivery of the drug to Tmab-resistant HER2-overexpressing breast cancer cells. These nanoparticles may pave the way for the development of novel therapies for poor prognosis resistant patients.

Intranasal immunization of lambs with serine/threonine phosphatase 2A against gastrointestinal nematodes.

Seven 3-month-old, female, helminth-free lambs were immunized intranasally with three doses (1 mg total) of a recombinant part of the catalytic region of the serine/threonine phosphatase 2A (PP2Ar) (group 1 [G1]). In addition, four lambs were used as an adjuvant control group (G2), four as unimmunized, infected controls (G3), and four as unimmunized, uninfected controls (G4). Fifteen days after the last immunization, lambs from G1, G2, and G3 were challenged with 10,000 larval stage 3 (L3) organisms in a plurispecific nematode infection composed of ca. 40% Trichostrongylus colubriformis, 40% Haemonchus contortus, and 20% Teladorsagia circumcincta. All the lambs were clinically monitored throughout the experiment. Parasitological (fecal egg output and immunological response), biopathological (packed-cell volume and leukocyte and eosinophil counts), and zootechnical (live-weight gain) analyses were conducted. On day 105 of the experiment, all the animals were slaughtered and the adult worm population in their abomasa examined. Intranasal administration of PP2Ar with bacterial walls as an adjuvant elicited a strong immune response in the immunized lambs, as evidenced by their humoral immune response. Immunized animals and animals receiving the adjuvant shed significantly (P < 0.001) fewer numbers of parasites' eggs in their feces. The immunization significantly reduced the helminth burden in the abomasa by the end of the experiment (>68%), protection being provided against both Haemonchus and Teladorsagia. Live-weight gain in the immunized lambs was similar to that in the uninfected controls versus the infected or adjuvanted animal groups. Our results suggest that heterologous immunization of ruminants by intranasal administration may be efficacious in the struggle to control gastrointestinal helminths in these livestock.