Alireza Zahedi

Cryptosporidium in humans and animals—a one health approach to prophylaxis

Cryptosporidium is a major cause of moderate‐to‐severe diarrhoea in humans worldwide, second only to rotavirus. Due to the wide host range and environmental persistence of this parasite, cryptosporidiosis can be zoonotic and associated with foodborne and waterborne outbreaks. Currently, 31 species are recognized as valid, and of these, Cryptosporidium hominis and Cryptosporidium parvum are responsible for the majority of infections in humans. The immune status of the host, both innate and adaptive immunity, has a major impact on the severity of the disease and its prognosis. Immunocompetent individuals typically experience self‐limiting diarrhoea and transient gastroenteritis lasting up to 2 weeks and recover without treatment, suggesting an efficient host antiparasite immune response. Immunocompromised individuals can suffer from intractable diarrhoea, which can be fatal. Effective drug treatments and vaccines are not yet available. As a result of this, the close cooperation and interaction between veterinarians, health physicians, environmental managers and public health operators is essential to properly control this disease. This review focuses on a One Health approach to prophylaxis, including the importance of understanding transmission routes for zoonotic Cryptosporidium species, improved sanitation and better risk management, improved detection, diagnosis and treatment and the prospect of an effective anticryptosporidial vaccine.

Public health significance of zoonotic Cryptosporidium species in wildlife: critical insights into better drinking water management

Cryptosporidium is an enteric parasite that is transmitted via the faecal–oral route, water and food. Humans, wildlife and domestic livestock all potentially contribute Cryptosporidium to surface waters. Human encroachment into natural ecosystems has led to an increase in interactions between humans, domestic animals and wildlife populations. Increasing numbers of zoonotic diseases and spill over/back of zoonotic pathogens is a consequence of this anthropogenic disturbance. Drinking water catchments and water reservoir areas have been at the front line of this conflict as they can be easily contaminated by zoonotic waterborne pathogens. Therefore, the epidemiology of zoonotic species of Cryptosporidium in free-ranging and captive wildlife is of increasing importance. This review focuses on zoonotic Cryptosporidium species reported in global wildlife populations to date, and highlights their significance for public health and the water industry.

Zoonotic Cryptosporidium species in animals inhabiting Sydney water catchments

Cryptosporidium is one of the most common zoonotic waterborne parasitic diseases worldwide and represents a major public health concern of water utilities in developed nations. As animals in catchments can shed human-infectious Cryptosporidium oocysts, determining the potential role of animals in dissemination of zoonotic Cryptosporidium to drinking water sources is crucial. In the present study, a total of 952 animal faecal samples from four dominant species (kangaroos, rabbits, cattle and sheep) inhabiting Sydney’s drinking water catchments were screened for the presence of Cryptosporidium using a quantitative PCR (qPCR) and positives sequenced at multiple loci. Cryptosporidium species were detected in 3.6% (21/576) of kangaroos, 7.0% (10/142) of cattle, 2.3% (3/128) of sheep and 13.2% (14/106) of rabbit samples screened. Sequence analysis of a region of the 18S rRNA locus identified C. macropodum and C. hominis in 4 and 17 isolates from kangaroos respectively, C. hominis and C. parvum in 6 and 4 isolates respectively each from cattle, C. ubiquitum in 3 isolates from sheep and C. cuniculus in 14 isolates from rabbits. All the Cryptosporidium species identified were zoonotic species with the exception of C. macropodum. Subtyping using the 5’ half of gp60 identified C. hominis IbA10G2 (n = 12) and IdA15G1 (n = 2) in kangaroo faecal samples; C. hominis IbA10G2 (n = 4) and C. parvum IIaA18G3R1 (n = 4) in cattle faecal samples, C. ubiquitum subtype XIIa (n = 1) in sheep and C. cuniculus VbA23 (n = 9) in rabbits. Additional analysis of a subset of samples using primers targeting conserved regions of the MIC1 gene and the 3’ end of gp60 suggests that the C. hominis detected in these animals represent substantial variants that failed to amplify as expected. The significance of this finding requires further investigation but might be reflective of the ability of this C. hominis variant to infect animals. The finding of zoonotic Cryptosporidium species in these animals may have important implications for the management of drinking water catchments to minimize risk to public health.

Cryptosporidium homai n. sp. (Apicomplexa: Cryptosporidiiae) from the guinea pig ( Cavia porcellus )

The morphological, biological, and molecular characterisation of a new Cryptosporidium species from the guinea pig (Cavia porcellus) are described, and the species name Cryptosporidium homai n. sp. is proposed. Histological analysis conducted on a post-mortem sample from a guinea pig euthanised due to respiratory distress, identified developmental stages of C. homai n. sp. (trophozoites and meronts) along the intestinal epithelium. Molecular analysis at 18S rRNA (18S), actin and hsp70 loci was then conducted on faeces from an additional 7 guinea pigs positive for C. homai n. sp. At the 18S, actin and hsp70 loci, C. homai n. sp. exhibited genetic distances ranging from 3.1% to 14.3%, 14.4% to 24.5%, and 6.6% to 20.9% from other Cryptosporidium spp., respectively. At the 18S locus, C. homai n. sp. shared 99.1% similarity with a previously described Cryptosporidium genotype in guinea pigs from Brazil and it is likely that they are the same species, however this cannot be confirmed as actin and hsp70 sequences from the Brazilian guinea pig genotype are not available. Phylogenetic analysis of concatenated 18S, actin and hsp70 sequences showed that C. homai n. sp. exhibited 9.1% to 17.3% genetic distance from all other Cryptosporidium spp. This clearly supports the validity of C. homai n. sp. as a separate species.

Molecular typing of Giardia duodenalis in humans in Queensland – first report of Assemblage E

Little is known about the genetic diversity of the protozoan parasite, Giardia duodenalis, infecting humans in Queensland, Australia. The present study typed 88 microscopically Giardia-positive isolates using assemblage-specific primers at the triose phosphate isomerase (tpi) gene and sequenced a subset of isolates at the glutamate dehydrogenase (gdh) gene (n = 30) and tpi locus (n = 27). Using the tpi-assemblage specific primers, G. duodenalis assemblage A and assemblage B were detected in 50% (44/88) and 38·6% (34/88) of samples, respectively. Mixed infections with assemblages A and B were identified in 4·5% (4/88) and assemblage E was identified in 6·8% (6/88) of samples. Sequence analysis at the gdh and tpi loci also confirmed the presence of assemblage E in these isolates. Cyst numbers per gram of feces (g-1) were determined using quantitative polymerase chain reaction and of the isolates that were typed as assemblage E, cyst numbers ranged 13·8-68·3 × 106 cysts g-1. This is the first report of assemblage E in humans in Australia, indicating that in certain settings, this assemblage may be zoonotic.

Next Generation Sequencing uncovers within-host differences in the genetic diversity of Cryptosporidium gp60 subtypes

The extent of within-host genetic diversity of parasites has implications for our understanding of the epidemiology, disease severity and evolution of parasite virulence. As with many other species, our understanding of the within-host diversity of the enteric parasite Cryptosporidium is changing. The present study compared Sanger and Next Generation Sequencing of glycoprotein 60 (gp60) amplicons from Cryptosporidium hominis (n=11), Cryptosporidium parvum (n=22) and Cryptosporidium cuniculus (n=8) DNA samples from Australia and China. Sanger sequencing identified only one gp60 subtype in each DNA sample: one C. hominis subtype (IbA10G2) (n=11), four C. parvum subtypes belonging to IIa (n=3) and IId (n=19) and one C. cuniculus subtype (VbA23) (n=8). Next Generation Sequencing identified the same subtypes initially identified by Sanger sequencing, but also identified additional gp60 subtypes in C. parvum and C. cuniculus but not in C. hominis, DNA samples. The number of C. parvum and C. cuniculus subtypes identified by Next Generation Sequencing within individual DNA samples ranged from two to four, and both C. parvum IIa and IId subtype families were identified within the one host in two samples. The finding of the present study has important implications for Cryptosporidium transmission tracking as well as vaccine and drug studies.

Prevalence of Cryptosporidium species and subtypes in paediatric oncology and non-oncology patients with diarrhoea in Jordan

Cryptosporidiosis is a protozoan parasitic disease which affects human and animals worldwide. In adult immunocompetent individuals, cryptosporidiosis usually results in acute and self-limited diarrhoea; however, it can cause life threatening diarrhoea in children and immunocompromised individuals. In the present study, we compared the prevalence of Cryptosporidium species and gp60 subtypes amongst paediatric oncology patients with diarrhoea (n=160) from King Hussein Medical Centre for Cancer in Jordan, and non-oncology paediatric patients with diarrhoea (n=137) from Al-Mafraq paediatric hospital. Microscopy results using modified acid fast staining identified a significantly (p≤0.05) higher prevalence of Cryptosporidium in paediatric oncology patients with diarrhoea (14.4% - 23/160), compared to non-oncology paediatric patients with diarrhoea only (5.1% - 7/137). With the exception of one sample, all microscopy-positive samples (n=29) and an additional 3/30 microscopy-negative controls were typed to species and subtype level at the 18S and gp60 loci, respectively. All Cryptosporidium positives were typed as C. parvum. Of the 22 typed Cryptosporidium positives from the paediatric oncology patients, 21 were subtyped as IIaA17G2R1 and one as IIaA16G2R1 C. parvum subtypes. The 7 typed positives from the paediatric patients from Al-Mafraq hospital were subtyped as IIaA17G2R1 (n=5) and IIaA16G2R1 (n=2). The 3 additional positives from the 30 microscopy negative control samples were subtyped as IIaA17G2R1. The high prevalence of the IIaA17G2R1 subtype, particularly amongst oncology patients, suggests that an outbreak of cryptosporidiosis may have been occurring in oncology patients during the collection period (April to December, 2016). New therapies for cryptosporidiosis in immunocompromised patients are urgently required.

Endemic, exotic and novel apicomplexan parasites detected during a national study of ticks from companion animals in Australia

BackgroundApicomplexan tick-borne pathogens that cause disease in companion animals include species of Babesia Starcovici, 1893, Cytauxzoon Neitz & Thomas, 1948, Hepatozoon Miller, 1908 and Theileria Bettencourt, Franca & Borges, 1907. The only apicomplexan tick-borne disease of companion animals that is known to occur in Australia is babesiosis, caused by Babesia canis vogeli Reichenow, 1937 and Babesia gibsoni Patton, 1910. However, no molecular investigations have widely investigated members of Apicomplexa Levine, 1980 in Australian ticks that parasitise dogs, cats or horses, until this present investigation.ResultsTicks (n = 711) removed from dogs (n = 498), cats (n = 139) and horses (n = 74) throughout Australia were screened for piroplasms and Hepatozoon spp. using conventional PCR and Sanger sequencing. The tick-borne pathogen B. vogeli was identified in two Rhipicephalus sanguineus Latreille ticks from dogs residing in the Northern Territory and Queensland (QLD). Theileria orientalis Yakimov & Sudachenkov, 1931 genotype Ikeda was detected in three Haemaphysalis longicornis Neumann ticks from dogs in New South Wales. Unexpectedly, the exotic tick-borne pathogen Hepatozoon canis James, 1905 was identified in an Ixodes holocyclus Neumann tick from a dog in QLD. Eight novel piroplasm and Hepatozoon species were identified and described in native ticks and named as follows: Babesia lohae n. sp., Babesia mackerrasorum n. sp., Hepatozoon banethi n. sp., Hepatozoon ewingi n. sp., Theileria apogeana n. sp., Theileria palmeri n. sp., Theileria paparinii n. sp. and Theileria worthingtonorum n. sp. Additionally, a novel cf. Sarcocystidae sp. sequence was obtained from Ixodes tasmani Neumann but could not be confidently identified at the genus level.ConclusionsNovel species of parasites in ticks represent an unknown threat to the health of companion animals that are bitten by these native tick species. The vector potential of Australian ticks for the newly discovered apicomplexans needs to be assessed, and further clinical and molecular investigations of these parasites, particularly in blood samples from dogs, cats and horses, is required to determine their potential for pathogenicity.

Profiling the diversity of Cryptosporidium species and genotypes in wastewater treatment plants in Australia using next generation sequencing

Wastewater recycling is an increasingly popular option in worldwide to reduce pressure on water supplies due to population growth and climate change. Cryptosporidium spp. are among the most common parasites found in wastewater and understanding the prevalence of human-infectious species is essential for accurate quantitative microbial risk assessment (QMRA) and cost-effective management of wastewater. The present study conducted next generation sequencing (NGS) to determine the prevalence and diversity of Cryptosporidium species in 730 raw influent samples from 25 Australian wastewater treatment plants (WWTPs) across three states: New South Wales (NSW), Queensland (QLD) and Western Australia (WA), between 2014 and 2015. All samples were screened for the presence of Cryptosporidium at the 18S rRNA (18S) locus using quantitative PCR (qPCR), oocyst numbers were determined directly from the qPCR data using DNA standards calibrated by droplet digital PCR, and positives were characterized using NGS of 18S amplicons. Positives were also screened using C. parvum and C. hominis specific qPCRs. The overall Cryptosporidium prevalence was 11.4% (83/730): 14.3% (3/21) in NSW; 10.8% (51/470) in QLD; and 12.1% (29/239) in WA. A total of 17 Cryptosporidium species and six genotypes were detected by NGS. In NSW, C. hominis and Cryptosporidium rat genotype III were the most prevalent species (9.5% each). In QLD, C. galli, C. muris and C. parvum were the three most prevalent species (7.7%, 5.7%, and 4.5%, respectively), while in WA, C. meleagridis was the most prevalent species (6.3%). The oocyst load/Litre ranged from 70 to 18,055 oocysts/L (overall mean of 3426 oocysts/L: 4746 oocysts/L in NSW; 3578 oocysts/L in QLD; and 3292 oocysts/L in WA). NGS-based profiling demonstrated that Cryptosporidium is prevalent in the raw influent across Australia and revealed a large diversity of Cryptosporidium species and genotypes, which indicates the potential contribution of livestock, wildlife and birds to wastewater contamination.

First report of Cryptosporidium parvum in a dromedary camel calf from Western Australia

Cryptosporidium is an important enteric parasite that can contribute large numbers of infectious oocysts to drinking water catchments. As a result of its resistance to disinfectants including chlorine, it has been responsible for numerous waterborne outbreaks of gastroenteritis. Wildlife and livestock play an important role in the transmission of Cryptosporidium in the environment. Studies conducted outside Australia have indicated that camels may also play a role in the transmission of zoonotic species of Cryptosporidium. Despite Australia being home to the world’s largest camel herd, nothing is known about the prevalence and species of Cryptosporidium infecting camels in this country. In the present study, C. parvum was identified by PCR amplification and sequencing of a formalin-fixed intestinal tissue specimen from a one-week old dromedary camel (Camelus dromedarius). Subtyping analysis at the glycoprotein 60 (gp60) locus identified C. parvum subtype IIaA17G2R1, which is a common zoonotic subtype reported in humans and animals worldwide. Histopathological findings also confirmed the presence of large numbers of variably-sized (1–3 μm in diameter) circular basophilic protozoa - consistent with Cryptosporidium spp.- adherent to the mucosal surface and occasionally free within the lumen. Further analysis of the prevalence and species of Cryptosporidium in camel populations across Australia are essential to better understand their potential for contamination of drinking water catchments.