The global crisis of plastic pollution, particularly involving microplastics (MPs) and nanoplastics (NPs), has profound ecological implications. Birds, serving as bioindicators, are especially susceptible to these pollutants. This systematic review synthesizes the current research on the presence, distribution, and impact of MPs and NPs on avian species, alongside advancements in detection methodologies. MPs and NPs have been identified in over 200 bird species across 46 families, encompassing several ecosystems, from Antarctica to Labrador, including Australia, China, and South Europe. Seabirds such as penguins, gulls, and shearwaters exhibit a high burden of MPs in tissues and feces due to fishing debris, while terrestrial species face contamination from urban and agricultural sources. Depending on their composition, MPs can cause gastrointestinal damage, oxidative stress, and bioaccumulation of toxic chemicals, particularly polyethylene and polypropylene. However, challenges in detection persist due to methodological inconsistencies, though advances in spectroscopy and flow cytometry offer improved accuracy. Addressing this pollution is vital for bird conservation and ecosystem health, requiring international collaboration and standardized research protocols.

The intraspecific variability among Neospora caninum isolates in their in vitro behaviour and in vivo virulence has been widely studied. In particular, transcriptomic and proteomic analyses have shown a higher expression/abundance of specific genes/proteins in high-virulence isolates. Consequently, the dense granule protein NcGRA7 and the rhoptry protein NcROP40 were proposed as potential virulence factors. The objective of this study was to characterize the role of these proteins using CRISPR/Cas9 knockout (KO) parasites in a well-established pregnant BALB/c mouse model of N. caninum infection at midgestation. The deletion of NcGRA7 and NcROP40 was associated with a reduction of virulence, as infected dams displayed milder clinical signs, lower parasite burdens in the brain, and reduced mortality rates compared to those infected with the wild-type parasite (Nc-Spain7). Specifically, those infected with the NcGRA7 KO parasites displayed significantly milder clinical signs and a lower brain parasite burden. The median survival time of the pups from dams infected with the two KO parasites was significantly increased, but differences in neonatal mortality rates were not detected. Overall, the present study indicates that the disruption of NcGRA7 considerably impairs virulence in mice, while the impact of NcROP40 deletion was more modest. Further research is needed to understand the role of these virulence factors during N. caninum infection.

The rise of antimicrobial resistance (AMR) has become a critical health challenge. This, plus the antimicrobial discovery void, had led scientists to search for an effective alternative to antimicrobials. In this context, nanomaterials, such as graphene oxide (GO), a two-dimensional (2D) carbon molecule with oxidized functional groups, have been shown to interact physically and chemically with bacteria. Moreover, the addition of polyethylene glycol (PEG) to its surface enhances GO’s biocompatibility and water solubility, making it a promising candidate for biomedical applications. This study evaluates the antimicrobial efficacy of GO and its polyethylene glycol-modified form (GO-PEG) against Staphylococcus aureus, a bacterium responsible for numerous hospital-acquired and multidrug-resistant infections. After their production, both nanomaterials were characterized using various techniques to provide insight into their morphology, stability, and functional group composition. Then, the antimicrobial activity of GO and GO-PEG was assessed using the Müeller–Hinton broth microdilution method, determining the minimum inhibitory concentration (MIC) for S. aureus among ten different concentrations of both nanomaterials (from 0.0625 to 32 mg/mL). The results demonstrate the potential of GO as an effective antimicrobial agent at 16 and 32 mg/mL, offering new strategies in the fight against AMR. Further research could establish its role in future therapeutic applications.

As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in Neospora caninum, an apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple mdhfr-ts copies into other sites of the genome. To determine the number of integrated mdhfr-ts in N. caninum, a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy wtdhfrs-ts gene, as well as the mutated mdhfrs-ts present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic NC5 gene. Thus, the dhfr-ts to NC5 ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated mdhrf-ts gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the Neospora KO strains NcΔGRA7 and NcΔROP40. For NcΔGRA7, the number of tachyzoites determined by dhfr-ts quantification was twice the number of tachyzoites determined by NC5 quantification, thus indicating that only one mdhfr-ts copy was integrated. The results obtained with the NcΔROP40 strain, however, showed that the number of dhfr-ts copies per genome was substantially higher, indicating that at least three copies of the selectable mdhfr-ts marker were integrated into the genomic DNA during gene editing by CRISPR-Cas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT N. caninum strains.

Neospora caninum is an apicomplexan parasite that causes abortion and stillbirth in cattle. We employed the pregnant neosporosis mouse model to investigate the efficacy of a modified version of the attenuated Listeria monocytogenes vaccine vector Lm3Dx_NcSAG1, which expresses the major N. caninum surface antigen SAG1. Multivalent vaccines were generated by the insertion of gra7 and/or rop2 genes into Lm3Dx_NcSAG1, resulting in the double mutants, Lm3Dx_NcSAG1_NcGRA7 and Lm3Dx_NcSAG1_NcROP2, and the triple mutant, Lm3Dx_NcSAG1_NcGRA7_NcROP2. Six experimental groups of female BALB/c mice were inoculated intramuscularly three times at two-week intervals with 1 × 10⁷ CFU of the respective vaccine strains. Seven days post-mating, mice were challenged by the subcutaneous injection of 1 × 10⁵N. caninum NcSpain-7 tachyzoites. Non-pregnant mice, dams and their offspring were observed daily until day 25 post-partum. Immunization with Lm3Dx_NcSAG1 and Lm3Dx_NcSAG1_NcGRA7_NcROP2 resulted in 70% postnatal pup survival, whereas only 50% and 58% of pups survived in the double mutant-vaccinated groups. Almost all pups had died at the end of the experiment in the infection control. The triple mutant was the most promising vaccine candidate, providing the highest rate of protection against vertical transmission (65%) and CNS infection. Overall, integrating multiple antigens into Lm3Dx_SAG1 resulted in lower vertical transmission and enhanced protection against cerebral infection in dams and in non-pregnant mice.