Cryptosporidium is a major cause of severe diarrhea-related disease in children in developing countries, but currently no vaccine or effective treatment exists for those who are most at risk of serious illness. This is partly due to the lack of in vitro culturing methods that are able to support the entire Cryptosporidium life cycle, which has led to research in Cryptosporidium biology lagging behind other protozoan parasites. In vivo models such as gnotobiotic piglets are complex, and standard in vitro culturing methods in transformed cell lines, such as HCT-8 cells, have not been able to fully support fertilization occurring in vitro. Additionally, the Cryptosporidium life cycle has also been reported to occur in the absence of host cells. Recently developed bioengineered intestinal models, however, have shown more promising results and are able to reproduce a whole cycle of infectivity in one model system. This review evaluates the recent advances in Cryptosporidium culturing techniques and proposes future directions for research that may build upon these successes.

Globally, gulls have been associated with carriage of high levels Escherichia coli resistant to critically important antimicrobial (CIA), a major concern as these antimicrobials are the sole or only one alternative amongst few available to treat severe life-threatening infections in humans. Previous study of Australian Silver Gulls demonstrated high levels of resistance to CIAs particularly fluoroquinolone and extended spectrum cephalosporin among E. coli (carriage 24% and 22% respectively). This study was aimed to identify and characterise strains from four distinct bird species inhabiting a common coastal environment, determine the frequency of carriage of CIA-resistant E. coli, and examine if these resistant clones and their resistance-encoding mobile genetic elements could be transmitted between species. CIA-resistant E. coli was detected in Silver Gulls (53%), Little Penguins (11%) and feral pigeons (10%), but not in Bridled Terns. In total, 37 different sequence types were identified including clinically significant human-associated lineages such as ST131, ST95, ST648, ST69, ST540, ST93, ST450 and ST10. Five main mobile genetic elements associated with blaCTX-M-positive E. coli isolated from three bird species were detected. Examination of clonal lineages and mobile genetic elements (MGEs) provided indirect evidence of transfer of resistance between bird species. The carriage of CIA-resistant E. coli by gulls and pigeons sharing proximity with humans, and in some instance food-producing animals increases the likelihood of further bi-directional dissemination. IMPORTANCE It has been shown that 20% of Australian Silver Gulls carry drug resistant Escherichia coli of anthropogenic origin associated with severe diseases such as sepsis and urinary tract infections in humans. To further characterise the dynamics of drug resistant E. coli in wildlife populations, we investigated the carriage of critically important antimicrobial (CIA) drug resistant E. coli in four bird species in a common environment. Our results indicate that gulls, pigeons and penguins carried drug resistant E. coli, and analysis of mobile genetic elements associated with resistance genes indicated interspecies resistance transfer. Terns, a bird species that forage on natural food sources at sea and distant from human, did not test positive for drug resistant E. coli. This study demonstrates carriage of CIA-resistant bacteria in multiple bird species inhabiting areas commonly inhabited by humans, and provides further evidence for a leap-frog effect of resistance between wildlife, facilitated by feeding habits.

Whole-genome sequencing is essential to many facets of infectious disease research. However, technical limitations such as bias in coverage and tagmentation, and difficulties characterising genomic regions with extreme GC content have created significant obstacles in its use. Illumina has claimed that the recently released DNA Prep library preparation kit, formerly known as Nextera Flex, overcomes some of these limitations. This study aimed to assess bias in coverage, tagmentation, GC content, average fragment size distribution, and de novo assembly quality using both the Nextera XT and DNA Prep kits from Illumina. When performing whole-genome sequencing on Escherichia coli and where coverage bias is the main concern, the DNA Prep kit may provide higher quality results; though de novo assembly quality, tagmentation bias and GC content related bias are unlikely to improve. Based on these results, laboratories with existing workflows based on Nextera XT would see minor benefits in transitioning to the DNA Prep kit if they were primarily studying organisms with neutral GC content.