Trypanosoma cruzi in Bats (Chiroptera; Mammalia) from the Brazilian Atlantic Forest, São Paulo State.

The causative agent of Chagas disease is Trypanosoma cruzi, which is widely distributed throughout the South American continent and extends into North America. Its occurrence in bats is poorly described and may impact the disease's maintenance and epidemiology. The aim of this study was to detect the agent by PCR assays targeting kDNA and nuclear DNA in the organs of 203 urban bats and rural vampire bats from the Brazilian Atlantic Forest, São Paulo state, during the pandemic period from 2020 to 2022. In total, 6 of the 203 bats (2.97%) were positive for T. cruzi. Infection was detected in 2% (2/101) of Desmodus rotundus, 33% (1/3) of Nyctinomops laticaudatus, 25% (1/4) of Artibeus lituratus, 4% (1/24) of Eumops glaucinus and in 2% (1/41) of Molossus molossus. The gene sequences obtained were assessed for quality and deposited in a public repository. Fruit bats were statistically associated with positivity for T. cruzi. To our knowledge, this study detected T. cruzi for the first time in bats from São Paulo state and in N. laticaudatus and E. glaucinus species.

Detection and molecular characterization of Cryptosporidium spp. in dairy calves in Lisbon and Tagus Valley, Portugal.

Cryptosporidium is a protozoan parasite with worldwide distribution, infecting a wide range of hosts with some zoonotic species. Calves have been identified as one of the most common reservoirs of this parasite. However, little is known about the genetics of Cryptosporidium in calves in Portugal. This study aimed to molecularly characterize infections of Cryptosporidium in pre-weaned calves from the Lisbon and Tagus Valley (LTV) in Portugal. Fifty-two samples were collected from calves from eight dairy and two beef farms in LTV, Portugal. Cryptosporidium oocysts were detected by Modified Ziehl-Neelsen staining (MZN) and direct immunofluorescent assay (DFA). MZN and DFA revealed the presence of Cryptosporidium oocysts in 40.4% (21/52) and 67.3% (35/52) samples, respectively. Positive samples were analyzed by PCR-RFLP of the 18 s rRNA gene for species identification. DNA amplification of the 18S rRNA gene was successful for 88.6% (31/35) of samples. Cryptosporidium parvum was identified in 96.8% (30/31) of the samples, and from one sample Cryptosporidium bovis was identified. Cryptosporidium parvum positive samples were subtyped by sequencing the PCR product of a partial fragment of the 60 kDa glycoprotein (gp60) gene. Subtype analysis of the C. parvum isolates revealed that all isolates belonged to subtype family IIa. Four subtypes were recognized within this subtype family, including the hyper-transmissible IIaA15G2R1 subtype that is the most frequently reported worldwide (27/30), IIaA14G2R1 (1/30), IIaA16G2R1 (1/30) and IIaA19G2R1 (1/30). To our knowledge, this is the first report of C. bovis, and C. parvum subtypes IIaA14G2R1 and IIaA19G2R1 in cattle in LTV, Portugal. The presence of the zoonotic C. parvum subtype in this study suggests that pre-weaned calves are likely to be a significant reservoir of zoonotic C. parvum, highlighting the importance of animal-to-human infection transmission risk. Further molecular studies are required to better understand the epidemiology of cryptosporidiosis in Portugal.

Avian Malaria in wild birds from a wildlife rehabilitation center in Central Portugal.

Avian haemosporidian (Haemoproteus, Leucocytozoon, Plasmodium) are vector-transmitted protozoan parasites highly prevalent in various bird species. Still, their importance for bird health, species decline, or impact on rehabilitation success is underestimated. This study aimed to determine the occurrence and diversity of haemosporidian parasites after necropsies of seventy wild birds from thirty-four species of twelve taxonomic orders. Detection of avian haemosporidian DNA was evaluated using PCR amplification of the cytochrome b gene. 48.6% of all sampled birds were positive, with 24.3% positive for Plasmodium spp./Haemoproteus spp. and 44.3% for Leucocytozoon spp. Mixed infections corresponded to 20% of all tested birds. Sequencing of several selected samples revealed the infection of Plasmodium matutinum, Plasmodium relictum and different lineages of Leucocytozoon spp. This study provides a baseline description of haemosporidian infections in wild birds from a rehabilitation center in central Portugal. The results show the necessity to test and monitor possible infections that undermine recovery processes for different birds. Further research into the occurrence of these haemosporidian species in birds kept in conservation centers is needed to understand the impact on bird health.

Isolation of saprophytic filamentous fungi from avian fecal samples and assessment of its predatory activity on coccidian oocysts.

Fungal strains used in the biocontrol of animal gastrointestinal parasites have been mainly isolated from pasture soil, decaying organic matter, and feces from herbivores and carnivores. However, their isolation from birds and assessment of predatory activity against avian GI parasites has been scarce thus far. This research aimed to isolate filamentous fungi from avian fecal samples and evaluate their predatory activity against coccidia. A pool of 58 fecal samples from chickens, laying hens, and peacocks, previously collected between July 2020-April 2021, were used for isolation of filamentous fungi and assessment of their in vitro predatory activity against coccidian oocysts, using Water-Agar medium and coprocultures. The Willis-flotation technique was also performed to obtain concentrated suspensions of oocysts. A total of seven Mucor isolates was obtained, being the only fungal taxa identified, and all presented lytic activity against coccidia. Isolates FR3, QP2 and SJ1 had significant coccidiostatic efficacies (inhibition of sporulation) higher than 70%, while isolates FR1, QP2 and QP1 had coccidicidal efficacies (destruction of the oocysts) of 22%, 14% and 8%, respectively, after 14 days of incubation, being a gradual and time-dependent process. To our knowledge, this is the first report regarding the isolation of native predatory fungi from avian feces and demonstration of their lytic activity against coccidia.

On the Occurrence and Multimerization of Two-Polypeptide Phage Endolysins Encoded in Single Genes.

Bacteriophages (phages) and other viruses are extremely efficient in packing their genetic information, with several described cases of overlapping genes encoded in different open reading frames (ORFs). While less frequently reported, specific cases exist in which two overlapping ORFs are in frame and share the stop codon. Here, we studied the occurrence of this genetic arrangement in endolysins, the phage enzymes that cut the bacterial cell wall peptidoglycan to release the virion progeny. After screening over 3,000 endolysin sequences of phages infecting Gram-positive bacteria, we found evidence that this coding strategy is frequent in endolysin genes. Our bioinformatics predictions were experimentally validated by demonstrating that two polypeptides are indeed produced from these genes. Additionally, we show that in some cases the two polypeptides need to interact and multimerize to generate the active endolysin. By studying in detail one selected example, we uncovered a heteromeric endolysin with a 1:5 subunit stoichiometry that has never been described before. Hence, we conclude that the occurrence of endolysin genes encoding two polypeptide isoforms by in-frame overlapping ORFs, as well as their organization as enzymatic complexes, appears more common than previously thought, therefore challenging the established view of endolysins being mostly formed by single, monomeric polypeptide chains. IMPORTANCE Bacteriophages use endolysins to cleave the host bacteria cell wall, a crucial event underlying cell lysis for virion progeny release. These bacteriolytic enzymes are generally thought to work as single, monomeric polypeptides, but a few examples have been described in which a single gene produces two endolysin isoforms. These are encoded by two in-frame overlapping ORFs, with a shorter ORF being defined by an internal translation start site. This work shows evidence that this endolysin coding strategy is frequent in phages infecting Gram-positive bacteria, and not just an eccentricity of a few phages. In one example studied in detail, we show that the two isoforms are inactive until they assemble to generate a multimeric active endolysin, with a 1:5 subunit stoichiometry never described before. This study challenges the established view of endolysins, with possible implications in their current exploration and design as alternative antibacterials.

Genetic variability, cryptic species and phylogenetic relationship of six cyathostomin species based on mitochondrial and nuclear sequences.

Cyathostomins are important intestinal nematode parasites of equines and include 50 accepted species. Their taxonomy has been frequently revised and the presence of cryptic species suggested. Furthermore, usually molecular- and morphology-based phylogenetic analyses give divergent results. In this study, the nucleotide sequences of the nuclear second internal transcribed spacer (ITS-2) and the mitochondrial partial cytochrome c oxidase subunit I (COI) were determined for adults of six cyathostomin species (Coronocyclus coronatus, Coronocyclus labiatus, Cylicocyclus nassatus, Cylicostephanus calicatus, Cylicostephanus longibursatus, Cylicostephanus minutus) collected from different equine species within two geographic regions. Maximum likelihood trees were calculated for ITS-2, COI, and concatenated data. No obvious differentiation was observed between geographic regions or equine host species. As previously reported, Coronocyclus coronatus and Cylicostephanus calicatus revealed a close relationship. Cryptic species were detected in Cylicostephanus minutus and Cylicostephanus calicatus. Cylicocyclus nassatus and Coronocyclus labiatus showed diverse mitochondrial and nuclear haplotypes occurring in different combinations, while Cylicostephanus longibursatus was comparatively homogenous. In conclusion, a combined analysis of nuclear and mitochondrial haplotypes improved resolution of the phylogeny and should be applied to the remaining cyathostomin species and across additional equine host species and geographic regions.

Nuclear and mitochondrial marker sequences reveal close relationship between Coronocyclus coronatus and a potential Cylicostephanus calicatus cryptic species complex.

The Cyathostominae (Nematoda, Strongyloidea) parasitising equines represent a diverse group currently including 50 species. However, their taxonomy has been repeatedly revised and occasionally the presence of cryptic genospecies was suggested. Moreover, molecular- and morphology-based phylogenetic analyses give divergent results. For instance, molecular data have suggested close relationship between Coronocyclus coronatus and Cylicostephanus calicatus, although morphology-based taxonomy places them in different genera. Here, nuclear (internal transcribed spacer 2, ITS-2) and mitochondrial (cytochrome oxidase I, COI) sequences were obtained from the same individual, morphologically identified worms. In both morphospecies, two ITS-2 sequences types were observed: In Cor. coronatus, a small PCR product of 278 bp (nuclear haplotype group nHGBco) was always present but often in combination with a larger 369-370 bp fragment (nHGAco). In Cys. calicatus, either a large 370 bp product (nHGAca) or a short 281 bp amplicon (nHGBca) were found, but never both. Sequence identity between morphospecies was up to 100%. The smaller differed from the larger fragments by deletion of the region 110-198 in Cor. coronatus and 112-203 in Cys. calicatus. In COI, three and five mitochondrial haplotype groups (HGs), mtHG1co-mtHG3co and mtHG1ca-mtHG5ca were identified for Cor. coronatus and Cys. calicatus, respectively. In Cor. coronatus, there was no particular association of mtHG with nuclear genotypes (only nHGBco vs. both nHGBco plus nHGAco). In Cys. calicatus the nHGAca was always associated with the mtHG1ca, mtHG2ca or mtHG5ca whereas nHGBca was exclusively associated with mtHG3ca or mtHG4ca. Despite up to 100% identity in the nHGs, no mixing of mtHGs was observed between both species. Clear separation of certain nHGs with particular mtHGs in Cys. calicatus, despite the fact that the same host individuals were infected with both groups simultaneously, suggests presence of two non-interbreeding genospecies within Cys. calicatus, which needs further confirmation using additional samples from diverse geographical origins.