Oocysts

The aim was to describe and analyse trends in the Cryptosporidium species and gp60 genotypes identified in human outbreaks of cryptosporidiosis in England and Wales from January 2009 to December 2017. Definitions used to define an outbreak were: an incident in which two or more people experienced a similar illness and linked in time or place, or a greater than expected rate of Cryptosporidium reports compared with the usual background rate for a place and time. Cryptosporidium outbreaks were extracted from the eFOSS database and from records for those that also came to the attention of the national CRU during outbreak investigations. The proportions of outbreak routes of transmission were compared between the two databases by uncorrected Chi square and a P-value of 0.05 was regarded as significant. The databases were reconciled by PHE centre, setting/place name, postcode, dates of first and last known cases, and populated with Cryptosporidium species and gp60 subtypes identified in the stools of cases and any additional samples tested. The outbreaks were analysed for trends in vehicles and settings, season, and associated Cryptosporidium species and gp60 subtypes. The CRU archive and the NCBI nucleotide DB and PubMed were searched for previous reports of subtypes found.
To identify species, Cryptosporidium positive stools were sent by primary diagnostic laboratories to the national CRU, generally within 5 days of collection [13 (link)]. Oocysts were separated from faecal material by salt flotation, disrupted by boiling, and DNA extracted using proteinase K digestion and a spin column kit (QIAamp DNA mini kit, Qiagen, Hilden, Germany) as described previously [13 (link)]. Samples were screened for C. parvum and C. hominis using a duplex real-time PCR assay [46 (link)] and other species were sought using a nested PCR targeting the SSU rDNA gene [47 (link)]. A nested PCR targeting the gp60 gene was used to subtype C. parvum and C. hominis samples known or suspected to be part of outbreaks as described previously [48 (link)]; to simplify workflow a cocktail of single round PCR primers was developed and used from 2015, as described previously [49 (link)]. PCR amplicons were subjected to bidirectional sequencing (Applied Biosystems 3500XL) and sequence similarities searched for in the NCBI Blastn website tools. Gp60 subtypes were confirmed by manual identification of trinucleotide repeats and other repeat sequences (Fig. 1). The findings were contextualised at the time to inform outbreak investigations and updated for this article.
In animal contact outbreaks, animals were sampled by a Veterinary Investigation Officer if requested by the outbreak control team and tested using immunofluorescence microscopy (Crypto-cel, Cellabs) at the Animal and Plant Health Agency’s central laboratory, Weybridge. Cryptosporidium-positive samples were sent to the CRU for genotyping as described above. In recreational and drinking water outbreaks, sampling and testing was undertaken as described in [7 ] if requested by the outbreak control team. Cryptosporidium positive microscope slides sent to the CRU for genotyping were processed as described previously [37 (link)] until 2015. After 2015 DNA extraction from slides was done using a chelex-based method as described previously [50 ].

This study used the coccidian parasite, Eimeria papillata, as a model parasite. Eimeria parasite oocysts were passaged in laboratory mice (Mus musculus). Unsporulated oocysts were collected from feces and allowed to sporulate in a solution containing 2.5% (w/v) potassium dichromate. They were then washed in phosphate buffer solution to prepare them for the remainder of the experiment.
Five groups of seven mice each were used, as follows: Group 1: Non-infected-non-treated (negative control). Group 2: Non-infected treated group with Bio-SeNPs (0.5 mg/kg of body weight). Groups 3-5 were orally inoculated with 1×10³ sporulated oocysts of E. papillata, according to Abdel-Tawab et al. (34 (link)). Group 3: Infected-non-treated (positive control). Group 4: Infected and treated group with Bio-SeNPs (0.5 mg/kg) (26 (link)). Group 5: Infected and treated group with the Amprolium (120 mg/kg body weight) (35 ). Groups 4 and 5 daily received oral administration (for 5 days) of Bio-SeNPs and anticoccidial medication, respectively, after 60 min of infection.
The Bio-SeNPs dose was chosen based on our preliminary experiment for determining the best dose inhibiting oocyst shed in mouse faeces (see Figure S1), as well as our previous work (26 (link)).
Weight change was assessed on day 0 and day 5 of the experiment, according to Al-Quraishy et al. (36 (link)). Fecal pellets from each mouse in the 3^rd, 4^th, and 5^th groups were collected on the 5^th day post-infection (p.i.), and the total number of shed oocysts was determined in accordance with Schito et al. (37 (link)). All the mice were sacrificed, and the jejuna were harvested and kept for use in the experiment’s subsequent stages.

Albino mice (BALB/c) were provided by the Animal Care Resource Center (ACRC) of the National Centre for Biological Sciences (NCBS), Bangalore, India. All animal procedures were approved and conducted according to the Instem IAEC under project INS-IAE-2019/01(N). The rodent malarial parasite P. berghei (ANKA strain-MRA-311, BEI Resources, USA) was used to infect mice by injection of 100–150 µl intraperitoneally per mouse [34 (link)]. Infected mice, upon reaching 5–6% parasitemia and 1:1 or 1:2 male:female gametocyte ratio, were anesthetized and exposed to 5–6-day-old female mosquitoes as detailed below. Prior to keeping mice for feeding mosquitoes, the final counts of parasitemia and exflagellation were recorded [34 (link)].
After eclosion, adult female mosquitoes were provided with 10% glucose solution for 4–6 days. Forty mosquitoes in two replicate cups were kept fasting for 6 h prior to blood feeding. The mosquito cups were covered with a black cloth and left undisturbed for 30–40 min during blood feeding. The fully engorged females were separated from unfed and half-fed mosquitoes. The cups were maintained in a bioenvironmental chamber (Percival insect chamber I-30VL, Perry, IA, USA) at 19 °C and 75% RH (12:12 h day and night cycles) for 13–14 days. A cotton wool pad was soaked with 10% d-glucose; 0.05% para-aminobenzoic acid (PABA) solution was changed every alternate day until dissection. The mosquito midguts were dissected on the 14th day using PBS and stained with 1% mercurochrome. Midguts were examined for the presence of oocysts and recorded under a microscope (Nikon eclipse Ni-U upright microscope, Japan).