Minimal zoonotic risk of cryptosporidiosis and giardiasis from frogs and reptiles.

The zoonotic potential of the protist parasites Cryptosporidium spp. and Giardia duodenalis in amphibians and reptiles raises public health concerns due to their growing popularity as pets. This review examines the prevalence and diversity of these parasites in wild and captive amphibians and reptiles to better understand the zoonotic risk. Research on Giardia in both groups is limited, and zoonotic forms of Cryptosporidium or Giardia have not been reported in amphibians. Host-adapted Cryptosporidium species dominate in reptiles, albeit some reptiles have been found to carry zoonotic (C. hominis and C. parvum) and rodent-associated (C. tyzzeri, C. muris and C. andersoni) species, primarily through mechanical carriage. Similarly, the limited reports of Giardia duodenalis (assemblages A, B and E) in reptiles may also be due to mechanical carriage. Thus, the available evidence indicates minimal zoonotic risk associated with these organisms in wild and captive frogs and reptiles. The exact transmission routes for these infections within reptile populations remain poorly understood, particularly regarding the importance of mechanical carriage. Although the risk appears minimal, continued research and surveillance efforts are necessary to gain a more comprehensive understanding of the transmission dynamics and ultimately improve our ability to safeguard human and animal health.

Rabbits as reservoirs: An updated perspective of the zoonotic risk from Cryptosporidium and Giardia.

Rabbits are highly abundant in many countries and can serve as reservoirs of diseases for a diversity of pathogens including the enteric protozoan parasites, Cryptosporidium and Giardia. Both parasites shed environmentally robust environmental stages (oo/cysts) and have been responsible for numerous waterborne outbreaks of diseases. Cryptosporidium hominis and C. parvum are responsible for most infections in humans, while Giardia duodenalis assemblages A and B, cause most human cases of giardiasis. Cryptosporidium cuniculus, the dominant species infecting rabbits, is the only spceies other than C. hominis and C. parvum to have caused a waterborne outbreak of gastritis, which occurred in the United Kingdom in 2008. This review examines the prevalence of Cryptosporidium and Giardia species in rabbits to better understand the public health risks of contamination of water sources with Cryptosporidium and Giardia oo/cysts from rabbits. Despite the abundance of C. cuniculus in rabbits, reports in humans are relatively rare, with the exception of the United Kingdom and New Zealand, and reports of C. cuniculus in humans from the United Kingdom have declined substantially since the 2008 outbreak. Subtyping of C. cuniculus has supported the potential for zoonotic transmission. Relatively few studies have been conducted on Giardia, but assemblage B dominates. However, improved typing methods are required to better understand the transmission dynamics of Giardia assemblages in rabbits. Similarly, it is not well understood if pet rabbits or contaminated water are the main source of C. cuniculus infections in humans. Well-planned studies using high-resolution typing tools are required to understand the transmission dynamics better and quantify the public health risk of Cryptosporidium and Giardia from rabbits.

Critters and contamination: Zoonotic protozoans in urban rodents and water quality.

Rodents represent the single largest group within mammals and host a diverse array of zoonotic pathogens. Urbanisation impacts wild mammals, including rodents, leading to habitat loss but also providing new resources. Urban-adapted (synanthropic) rodents, such as the brown rat (R. norvegicus), black rat (R. rattus), and house mouse (Mus musculus), have long successfully adapted to living close to humans and are known carriers of zoonotic pathogens. Two important enteric, zoonotic protozoan parasites, carried by rodents, include Cryptosporidium and Giardia. Their environmental stages (oocysts/cysts), released in faeces, can contaminate surface and wastewaters, are resistant to common drinking water disinfectants and can cause water-borne related gastritis outbreaks. At least 48 species of Cryptosporidium have been described, with C. hominis and C. parvum responsible for the majority of human infections, while Giardia duodenalis assemblages A and B are the main human-infectious assemblages. Molecular characterisation is crucial to assess the public health risk linked to rodent-related water contamination due to morphological overlap between species. This review explores the global molecular diversity of these parasites in rodents, with a focus on evaluating the zoonotic risk from contamination of water and wasterwater with Cryptosporidium and Giardia oocysts/cysts from synanthropic rodents. Analysis indicates that while zoonotic Cryptosporidium and Giardia are prevalent in farmed and pet rodents, host-specific Cryptosporidium and Giardia species dominate in urban adapted rodents, and therefore the risks posed by these rodents in the transmission of zoonotic Cryptosporidium and Giardia are relatively low. Many knowledge gaps remain however, and therefore understanding the intricate dynamics of these parasites in rodent populations is essential for managing their impact on human health and water quality. This knowledge can inform strategies to reduce disease transmission and ensure safe drinking water in urban and peri­urban areas.

Zoonotic Cryptosporidium and Giardia in marsupials-an update.

Marsupials, inhabiting diverse ecosystems, including urban and peri-urban regions in Australasia and the Americas, intersect with human activities, leading to zoonotic spill-over and anthroponotic spill-back of pathogens, including Cryptosporidium and Giardia. This review assesses the current knowledge on the diversity of Cryptosporidium and Giardia species in marsupials, focusing on the potential zoonotic risks. Cryptosporidium fayeri and C. macropodum are the dominant species in marsupials, while in possums, the host-specific possum genotype dominates. Of these three species/genotypes, only C. fayeri has been identified in two humans and the zoonotic risk is considered low. Generally, oocyst shedding in marsupials is low, further supporting a low transmission risk. However, there is some evidence of spill-back of C. hominis into kangaroo populations, which requires continued monitoring. Although C. hominis does not appear to be established in small marsupials like possums, comprehensive screening and analysis are essential for a better understanding of the prevalence and potential establishment of zoonotic Cryptosporidium species in small marsupials. Both host-specific and zoonotic Giardia species have been identified in marsupials. The dominance of zoonotic G. duodenalis assemblages A and B in marsupials may result from spill-back from livestock and humans and it is not yet understood if these are transient or established infections. Future studies using multilocus typing tools and whole-genome sequencing are required for a better understanding of the zoonotic risk from Giardia infections in marsupials. Moreover, much more extensive screening of a wider range of marsupial species, particularly in peri-urban areas, is required to provide a clearer understanding of the zoonotic risk of Cryptosporidium and Giardia in marsupials.

The risk of wild birds contaminating source water with zoonotic Cryptosporidium and Giardia is probably overestimated.

Cryptosporidium and Giardia are important waterborne protozoan parasites that are resistant to disinfectants commonly used for drinking water. Wild birds, especially wild migratory birds, are often implicated in the contamination of source and wastewater with zoonotic diseases, due to their abundance near water and in urban areas and their ability to spread enteric pathogens over long distances. This review summarises the diversity of Cryptosporidium and Giardia in birds, with a focus on zoonotic species, particularly in wild and migratory birds, which is critical for understanding zoonotic risks. The analysis revealed that both avian-adapted and zoonotic Cryptosporidium species have been identified in birds but that avian-adapted Cryptosporidium species dominate in wild migratory birds. Few studies have examined Giardia species and assemblages in birds, but the non-zoonotic Giardia psittaci and Giardia ardeae are the most commonly reported species. The identification of zoonotic Cryptosporidium and Giardia in birds, particularly C. parvum and G. duodenalis assemblages A and B in wild migratory birds, is likely due to mechanical carriage or spillback from birds co-grazing pastures contaminated with C. parvum from livestock. Therefore, the role of wild migratory birds in the transmission of zoonotic Cryptosporidium and Giardia to source water is likely overestimated. To address knowledge gaps, it is important to conduct more extensive studies on the prevalence of Cryptosporidium and Giardia in a broader range of migratory wild birds. There is also a need to investigate the extent to which zoonotic infections with C. hominis/C. parvum and G. duodenalis assemblages A and B are mechanical and/or transient, and to assess the load and viability of zoonotic oo/cysts shed in avian faeces. Understanding the contribution of birds to zoonoses is essential for effective disease surveillance, prevention, and control.

Cryptosporidium and Giardia in cats and dogs: What is the real zoonotic risk?

Due to the close bond between humans and companion animals, a thorough understanding of the diversity of Cryptosporidium species and Giardia assemblages in cats and dogs is essential to determine the potential zoonotic risks. Analysis of molecular studies shows that C. felis and C. canis are the main species infecting cats and dogs, respectively. These species are largely host-specific, as despite intense association with humans, prevalence of C. felis and C. canis in humans is low and predominantly in immunocompromised individuals and low-income countries. There have been reports of C. parvum in cats and dogs and two reports of C. hominis in dogs. In most studies conducted to date, however, the prevalence of zoonotic species was low and may be associated with coprophagy and or/spillback, but this remains to be determined. Results of subtyping studies suggest that for C. felis and C. canis, some zoonotic transmission may occur but host-adapted subtypes also exist. Giardia duodenalis assemblages C and D are commonly reported in dogs, with assemblages F and A most common in cats. Assemblages C, D and F are largely host-specific as there are only a handful of reports of them in humans. Reports of assemblage A and B in cats and dogs may be due to coprophagy or spillback from owners. Despite the extent of pet ownership and the close contact between humans and companion animals worldwide, the overall risk of zoonotic transmission from cats and dogs to humans is uncertain but thought to be low due to C. canis, C. felis and G. duodenalis assemblages C, D and F being predominantly host-specific, the relatively low prevalence of C. parvum (and C. hominis) in cats and dogs (which may be due to mechanical carriage), and low oo/cyst shedding. Carefully designed epidemiological studies of cats and dogs and their owners using subtyping tools are essential to better quantify the extent of spillover and spillback of Cryptosporidium and Giardia between pets and their owners.

How significant are bats as potential carriers of zoonotic Cryptosporidium and Giardia?

Bats are known to harbour various pathogens and are increasingly recognised as potential reservoirs for zoonotic diseases. This paper reviews the genetic diversity and zoonotic potential of Cryptosporidium and Giardia in bats. The risk of zoonotic transmission of Cryptosporidium from bats to humans appears low, with bat-specific Cryptosporidium genotypes accounting for 91.5% of Cryptosporidium-positive samples genotyped from bats worldwide, and C. parvum and C. hominis accounting for 3.4% each of typed positives, respectively. To date, there have only been sporadic detections of Giardia in bats, with no genetic characterisation of the parasite to species or assemblage level. Therefore, the role bats play as reservoirs of zoonotic Giardia spp. is unknown. To mitigate potential risks of zoonotic transmission and their public health implications, comprehensive research on Cryptosporidium and Giardia in bats is imperative. Future studies should encompass additional locations across the globe and a broader spectrum of bat species, with a focus on those adapted to urban environments.

Sertraline has fungicidal activity against Candida spp. and acts by inhibiting membrane and cell wall biosynthesis.

Aim: Our study evaluated the activity of sertraline (SER) alone and associated with antifungal drugs in planktonic Candida spp. strains, and investigated its mechanism of action. Materials & methods: Broth microdilution method and minimum fungicidal concentration/MIC ratio were used to assess SER anticandidal activity, and the interaction with antifungals was determined by fractional inhibitory concentration index. The mechanism of action was investigated by flow cytometry and in silico tests. Results: SER inhibited Candida spp. strains at low concentrations by the fungicidal effect and showed no loss of effectiveness when combined. Its action seemed to be related to the membrane and cell wall biosynthesis inhibition. Conclusion: SER has activity against Candida spp. isolated and associated with antifungals, and acts by causing cell wall and membrane damage.

Antifungal activity of cisatracurium against fluconazole-resistant Candida isolates and its antibiofilm effects.

Aim: To evaluate the antifungal activity of cisatracurium against Candida spp. resistant to fluconazole strains in planktonic and biofilm forms, in addition to determining its mechanism of action. Materials & methods: Antifungal activity and pharmacological interactions were determined using broth microdilution methods and the mechanism of action was evaluated by flow cytometry and molecular docking. Results: Cisatracurium presented antifungal activity against Candida spp. planktonic cells due to alterations of mitochondrial transmembrane potential leading to cellular apoptosis in addition to interacting with important targets related to cellular respiration, membrane and cell wall evidenced by molecular docking. Furthermore, the drug both prevented biofilm formation and impaired mature biofilms. Conclusion: Cisatracurium exhibits potential antifungal activity against Candida spp.

Activity of amlodipine against Staphylococcus aureus: association with oxacillin and mechanism of action.

Aim: This study was designed to evaluate the in vitro antimicrobial activity of amlodipine against Staphylococcus aureus strains. Materials & methods: The antimicrobial activity of amlodipine was evaluated by the broth microdilution method and its interaction with oxacillin was evaluated by checkerboard assay. The possible mechanism of action was evaluated by flow cytometry and molecular docking techniques. Results: Amlodipine showed activity against S. aureus between 64 and 128 µg/ml, in addition to showing synergism in approximately 58% of the strains used. Amlodipine also showed good activity against forming and mature biofilms. The possible mechanism of action may be attributed to its ability to lead to cell death. Conclusion: Amlodipine has antibacterial activity against S. aureus.

Antibacterial activity of paroxetine against Staphylococcus aureus and possible mechanisms of action.

Aim: To evaluate the antibacterial activity of paroxetine alone and associated with oxacillin against isolates of methicillin-sensitive and -resistant Staphylococcus aureus. Materials & methods: The broth microdilution and checkerboard techniques were used, with investigation of possible mechanisms of action through flow cytometry, fluorescence microscopy and molecular docking, in addition to scanning electron microscopy for morphological analysis. Results: Paroxetine showed a MIC of 64 µg/ml and bactericidal activity, mostly additive interactions in combination with oxacillin, evidence of action on genetic material and membrane, morphological changes in microbial cells and influence on virulence factors. Conclusion: Paroxetine has antibacterial potential from the perspective of drug repositioning.

Distribution and Prevalence of Theileria orientalis Genotypes in Adult Lactating Dairy Cows in South West Region of Western Australia.

Bovine anaemia caused by Theileria orientalis group (BATOG) causes significant production and economic losses in Australia's cattle industry. The pathogenic T. orientalis genotypes reported in Australian cattle are type 1 (Chitose) and type 2 (Ikeda). The present study aimed to determine the prevalence and distribution of T. orientalis genotypes in adult lactating cows in Western Australia (WA) dairy herds. A total of 100 whole blood samples from lactating cows from 10 farms were obtained and screened for T. orientalis using polymerase chain reaction (PCR). Sanger sequencing was subsequently used to characterise T. orientalis genotypes isolated from positive samples. A total of thirteen cows (13%; 95% CI: 7.1-21.2%) were positive for T. orientalis, and six out of ten farms (60%; 95% CI: 26.2-87.8%) housed at least one T. orientalis-positive cow. The distribution of T. orientalis was found to be wide and dense in the South west region of WA and the southern coast of WA. The predominant T. orientalis genotype identified was Ikeda (n = 11, 11%; 95% CI: 5.6-18.8%), while the Buffeli genotype was identified in WA for the first time, albeit at a low prevalence (n = 1, 1%; 95% CI: 0.0-5.4%). This study has provided useful epidemiological evidence on the prevalence and distribution of T. orientalis in adult lactating dairy cows in WA dairy farms, and on the importance of conducting widespread surveillance programs for the understanding of BATOG in WA.

A systems biology approach to better understand human tick-borne diseases.

Tick-borne diseases (TBDs) are a growing global health concern. Despite extensive studies, ill-defined tick-associated pathologies remain with unknown aetiologies. Human immunological responses after tick bite, and inter-individual variations of immune-response phenotypes, are not well characterised. Current reductive experimental methodologies limit our understanding of more complex tick-associated illness, which results from the interactions between the host, tick, and microbes. An unbiased, systems-level integration of clinical metadata and biological host data - obtained via transcriptomics, proteomics, and metabolomics - offers to drive the data-informed generation of testable hypotheses in TBDs. Advanced computational tools have rendered meaningful analysis of such large data sets feasible. This review highlights the advantages of integrative system biology approaches as essential for understanding the complex pathobiology of TBDs.

The Troublesome Ticks Research Protocol: Developing a Comprehensive, Multidiscipline Research Plan for Investigating Human Tick-Associated Disease in Australia.

In Australia, there is a paucity of data about the extent and impact of zoonotic tick-related illnesses. Even less is understood about a multifaceted illness referred to as Debilitating Symptom Complexes Attributed to Ticks (DSCATT). Here, we describe a research plan for investigating the aetiology, pathophysiology, and clinical outcomes of human tick-associated disease in Australia. Our approach focuses on the transmission of potential pathogens and the immunological responses of the patient after a tick bite. The protocol is strengthened by prospective data collection, the recruitment of two external matched control groups, and sophisticated integrative data analysis which, collectively, will allow the robust demonstration of associations between a tick bite and the development of clinical and pathological abnormalities. Various laboratory analyses are performed including metagenomics to investigate the potential transmission of bacteria, protozoa and/or viruses during tick bite. In addition, multi-omics technology is applied to investigate links between host immune responses and potential infectious and non-infectious disease causations. Psychometric profiling is also used to investigate whether psychological attributes influence symptom development. This research will fill important knowledge gaps about tick-borne diseases. Ultimately, we hope the results will promote improved diagnostic outcomes, and inform the safe management and treatment of patients bitten by ticks in Australia.

Activity of arginine-phenylalanine and arginine-tryptophan-based surfactants against Staphylococcus aureus.

Aims: This study aimed to evaluate the antibacterial effect of two new cationic surfactants based on phenylalanine-arginine (LPAM) and tryptophan-arginine (LTAM). Materials & methods: Antibacterial activity, mechanism of action and interactions with Staphylococcus aureus enzymes were measured through microbiological, flow cytometry and molecular docking assays, respectively. Results & conclusion: These compounds showed antibacterial activity in the range of 4.06-16.24 µg/ml against planktonic cells and no activity against mature biofilms, since they caused a loss of membrane integrity and increased DNA damage, as revealed by flow cytometry analysis. In silico assays revealed the existence of molecular bonds such as hydrogen bonds, mainly with DNA. Therefore, these compounds have promising pharmacological activity against MRSA strains.

Synergistic activity of dobutamine combined with azoles and its mechanism of action against strains of Candida glabrata.

Aims: To evaluate the antifungal effect of dobutamine against Candida glabrata as well as its synergism with azoles and its action on biofilm. Methods: The M27-A3 protocol and flow cytometry were used for elucidation of the possible mechanism of action. Results: The tested isolates presented MICs ranging from 2 to 32 µg/ml for dobutamine, with fungistatic effect. A total of 82% of the strains showed synergism with fluconazole, with 90% showing synergism with itraconazole. The effect on biofilm formation was nonsignificant. Cytometry tests showed that dobutamine induced mitochondrial depolarization. Conclusion: Dobutamine has an antifungal effect on strains of C. glabrata and synergistic activity with azoles. This effect is probably mediated by increased oxidative damage to the membrane.

Diversity and Epidemiology of Bat Trypanosomes: A One Health Perspective.

Bats (order Chiroptera) have been increasingly recognised as important reservoir hosts for human and animal pathogens worldwide. In this context, molecular and microscopy-based investigations to date have revealed remarkably high diversity of Trypanosoma spp. harboured by bats, including species of recognised medical and veterinary importance such as Trypanosoma cruzi and Trypanosoma evansi (aetiological agents of Chagas disease and Surra, respectively). This review synthesises current knowledge on the diversity, taxonomy, evolution and epidemiology of bat trypanosomes based on both molecular studies and morphological records. In addition, we use a One Health approach to discuss the significance of bats as reservoirs (and putative vectors) of T. cruzi, with a focus on the complex associations between intra-specific genetic diversity and eco-epidemiology of T. cruzi in sylvatic and domestic ecosystems. This article also highlights current knowledge gaps on the biological implications of trypanosome co-infections in a single host, as well as the prevalence, vectors, life-cycle, host-range and clinical impact of most bat trypanosomes recorded to date. Continuous research efforts involving molecular surveillance of bat trypanosomes are required for improved disease prevention and control, mitigation of biosecurity risks and potential spill-over events, ultimately ensuring the health of humans, domestic animals and wildlife globally.

Evaluation of the antifungal activity in vitro of midazolam against fluconazole-resistant Candida spp. isolates.

Aim: The purpose of this study was to evaluate the antifungal activity of midazolam, alone and in association with azoles, against isolates of clinical Candida spp. in planktonic and biofilm form. Materials & methods: The antifungal activity was observed using the broth microdilution technique. Flow cytometry tests were performed to investigate the probable mechanism of action and the comet test and cytotoxicity test were applied to evaluate DNA damage. Results: Midazolam (MIDAZ) showed antifungal activity against planktonic cells (125-250 µg/ml) and reduced the viability of Candida spp. biofilms (125 a 2500 µg/ml). The interaction of MIDAZ against Candida spp. biofilms was observed through scanning electron microscopy, causing alteration of their appearance. Therefore, MIDAZ has antifungal potential against Candida spp.

First Report of Candidatus Mycoplasma haemohominis Infection in Australia Causing Persistent Fever in an Animal Carer.

BACKGROUND: Hemotropic mycoplasmas (hemoplasmas) infect animals and humans and can lead to clinical syndromes mainly characterized by hemolytic anemia. A novel pathogen, Candidatus Mycoplasma haemohominis, was recently associated with a case of human hemoplasmosis in Europe. Here we report the first detection of this pathogen in an Australian patient exhibiting persistent fever, hemolytic anemia, and pancytopenia over a 10-month period. METHODS: After exhaustive negative testing for human infectious diseases, whole genome sequencing (WGS) was performed on the patient's bone marrow aspirate, using an Illumina NextSeq500 platform. Conventional polymerase chain reaction (PCR), followed by Sanger sequencing, was then performed on blood samples using novel Mycoplasma-specific primers targeting the 16S ribosomal RNA gene. In addition, a Mycoplasma-specific fluorescence in situ hybridization (FISH) assay was developed to differentiate Mycoplasma cells from other erythrocyte inclusions (eg, Pappenheimer and Howell-Jolly bodies) which are morphologically similar to bacterial cocci by light microscopy. RESULTS: WGS analysis revealed that approximately 0.04% of the total number of unmapped reads to human genome corresponded to Mycoplasma species. A 1-kb Mycoplasma 16S fragment was successfully amplified by conventional PCR, and sequence analyses revealed 100% identity with Candidatus Mycoplasma haemohominis. FISH confirmed that several (approximately 2%) epierythrocytic inclusions initially observed by light microscopy corresponded to Mycoplasma cells. CONCLUSIONS: This represents the second report of hemolytic anemia associated with hemoplasma infection in a human, and the first report of human hemoplasmosis in Australia. This study highlights the importance of new and emerging diagnostic approaches and need for further investigations on the epidemiology of Candidatus Mycoplasma haemohominis in Australia.

Sequence analyses at mitochondrial and nuclear loci reveal a novel Theileria sp. and aid in the phylogenetic resolution of piroplasms from Australian marsupials and ticks.

The order Piroplasmida encompasses two main families: Babesiidae and Theileriidae, containing tick-borne pathogens of veterinary and medical importance worldwide. While only three genera (Babesia, Cytauxzoon and Theileria) comprising piroplasm parasites are currently recognised, phylogenetic studies at the 18S rRNA (18S) gene suggest that these organisms represent at least ten lineages, one of which comprises the relatively unique and highly diverse Theileria spp. from Australian marsupials and ticks. As an alternative to analysing 18S sequences alone, sequencing of mitochondrial genes has proven to be useful for the elucidation of evolutionary relationships amongst some groups of piroplasms. This research aimed to characterise piroplasms from Australian native mammals and ticks using multiple genetic markers (18S, cytochrome c, oxidase subunit III (cox3) and cytochrome B (cytB)) and microscopy. For this, nearly complete piroplasm-18S sequences were obtained from 32 animals belonging to six marsupial species: eastern bettong (Bettongia gaimardi), eastern quoll (Dasyurus viverrinus), eastern grey kangaroo (Macropus giganteus), swamp wallaby (Wallabia bicolor), quokka (Setonix brachyurus) and Gilbert's potoroo (Potorous gilbertii). The organisms detected represented eight novel Theileria genotypes, which formed five sub-clades within the main marsupial clade containing previously reported Australian marsupial and tick-derived Theileria spp. A selection of both novel and previously described Australian piroplasms at the 18S were also successfully characterised, for the first time, at the cox3 and cytB loci, and corroborated the position of Australian native theilerias in a separate, well-supported clade. Analyses of the cox3 and cytB genes also aided in the taxonomic resolution within the clade of Australian Piroplasmida. Importantly, microscopy and molecular analysis at multiple loci led to the discovery of a unique piroplasm species that clustered with the Australian marsupial theilerias, for which we propose the name Theileria lupei n. sp.