Microbiome diversity is a modifiable virulence factor for cryptosporidiosis

Figures & data

Table 1. Alpha diversity indexes and their corresponding equations.

	Diversity Index	Formula	Reference
Alpha Diversity Indexes	Shannon Diversity Index	$H=-\sum _{i=1}^s\left(p_i\mathrm{l}\mathrm{n}{p}_i\right)$	Lemos et al (2010) [22]
		s is the number of OTUs, pi is the proportion of the community represented ay OTU i.
	Shannon Reciprocal index	$\frac{1}{\mathrm{l}\mathrm{n}\left(H\right)}$
	Simpson Diversity Index	$D=\frac{1}{{\sum }_{i=1}^s{p}_i^2}$	Simpson (1949) [23]
		s is the total number of species in the community, pi is the proportion pf community represented by OTU i.
	Simpson’s Reciprocal Index	$\frac{1}{D}$

Figure 1. Overview of study selection process. PRISMA flow chart showing the article screening process from three databases: PubMed, Google Scholar, Web of Science.

Table 2. Compiled Shannon index scores (H) from studies further described in Table 3.

Author (Year)	Subject	Symptoms	Treatment	Group/Animal Name	Infection Status	Shannon Index (H)
Ras et al. (2015) [30]	Adult CD-1 outbred mice	Not recorded	Immunosuppressed – Dexamethasone	Group 1 – MD C. parvum isolate	Uninfected	4.9
					Infected	5.1
				Group 2 – TU114 C. parvum isolate	Uninfected	5.0
					Infected	4.8
Charania et al. (2020) [34]	IL-12 KO C57BL16 mice	Not recorded	No treatment for groups used in this review		Uninfected	4.0
					Infected	3.7
McKenney et al. (2017) [33]	Coquerel sifaka’s – Captivity	Lethargy, anorexia, or diarrhoea	1dpi – Antimicrobials 7dpi – Antibiotics Received faecal Transplant	Primate-1	Uninfected	6.7
					Infected	5.0
			3dpi – Antimicrobials 8dpi – Antibiotics Received Faecal Transplant	Primate-2	Uninfected	8.0
					Infected	6.2
			1dpi – Antimicrobials 6dpi – Antibiotics Received Faecal Transplant	Primate-3	Uninfected	8.1
					Infected	5.9
			No Treatment	Primate-4	Uninfected	8.8
					Infected	7.5
			11dpi - Antibiotics	Primate-5	Uninfected	7.9
					Infected	6.0
			No treatment	Primate-6	Uninfected	8.0
					Infected	7.7
Mammeri et al. (2019) [31]	5 day old, Outbred, CD-1 mice	Not recorded	No treatment mentioned	Experiment 1	Uninfected	0.78
					Infected	1.0
				Experiment 2 – Repeat of Experiment 1	Uninfected	0.95
					Infected	1.46
Mammeri et al. (2020) [32]	21 day old, male, French Alpine goat kids	Watery, yellow faeces with clumps and mucus. Hypothermia, dehydration, growth deformities, weight loss and death.	No treatment	Control	Uninfected	9.5
				Infected	Infected	7.0

Table 3. Studies included in this review.

Aim Add ressed in Review	Authors	Date of Study	Accession Numbers	Participants (number of subjects)	Clinical Symptoms	Intervention (Length of Treatment)	Prior Antimicrobial Treatment?	Effect on Oocyst Shedding
1	Ras et al [30]	2015	PRJEB7853	Adult out-bred CD-1 mice (30)	-	No intervention	Dexamethasone 21-phosphate (Day 8–2 days after first dose mice were challenged with C. parvum)	Study did not measure this outcome – focus was on bacterial diversity.
1	Mammeri et al [31]	2019	-	5-day old neonatal outbred CD-1 mice (36)	-	No intervention	No prior treatment	Study did not measure this outcome – focus was on bacterial diversity.
1	Mammeri et al [32]	2020	PRJNA603642	21 day old, male, French Alpine goat kids	Watery, yellow faeces with clumps and mucus. Hypothermia, dehydration, growth deformities, weight loss and death.	No intervention	No prior treatment	Study did not measure this outcome – focus was on bacterial diversity.
1 and 2	McKenney et al [33]	2017	SAMN06349027–SAMN06349170	Coquerel’s sifakas (35)	Lethargy, anorexia, or diarrhoea	Treatment varied between primates, listed are interventions used on the sifakas which received faecal transplants. Ampicillin (6 days), Ceftazidime (6 days), Metronidazole (5.5 days), nitazoxanide (12.6 days), ceftiofur (5 days) Faecal transplants originated from healthy donors.	None – Treatment given to specific animals during infection. See Table 2.	Faecal transplants resulted in resolution of symptoms. Diversity of faecal microbiome higher after transplant than pre-infection levels. Those without faecal transplants had reduced diversity after infection.
1 and 2	Charania et al [34]	2020	-	IL-12 KO C57BL/6 mice (NumberUnknown)	-	Cloxacillin (1 day prior to infection) Paromomycin (1 day prior to infection) Vancomycin-Imipenem (1 week prior to infection)	None	More oocysts shed in mice givencloxacillin, than vancomycin-imipenem Lower bacterial diversity in cloxacillin treated mice than vancomycin-imipenem and paromomycin treated mice.
2	Alak et al [35]	1997	-	C57BL/6 female mice (40)	Oocysts in faeces	Lactobacillus reuteri (10 days prior to infection)	LP-BM5 treatment	L. reuteri supplemented mice showed reduced oocyst shedding compared to controls
2	Pickerd and Tuthill [36]	2004	-	12-year old girl with Coeliac’s Disease (1)	4 months abdominal pain, flatulence, loose stools, nausea, lethargy.	Lactobacillus GG and Lactobacillus casei Shirota (4 weeks)	None	Diarrhoea and nausea resolved in 10 days
2	Oliveira and Widmer [37]	2018	PRJEB25162, PRJEB25164	CD-1 female mice (16)	-	Probiotics consisting of: Lactobacillus (9 species), Bifidobacterium (4 species), Streptococcus thermophilus, acacia gum, larch gum, galacto-oligosaccharide, L-glutamine, vitamin D3 (1 day prior to infection)	Dexamethasone 21-phosphate disodium (7 days prior to infection) Vancomycin and streptomycin (6 days prior to infection) Metronidazole (6 days prior to infection)	Mice given probiotics released 8 times more oocysts than control mice. No significant increase of probiotic bacteria in faeces.
2	Ichikawa-Seki et al [38]	2019	-	Neonatal Holstein calves, female (20)	Faecal score was measured. No additional symptoms listed.	No Intervention. Infections were naturally occurring.	None	Increase in Fusobacterium was associated with increase oocyst shedding, and an increase in faecal scoring.
2	Oliveira and Widmer [39]	2019	PRJEB31954, PRJEB31955, PRJEB31958, PRJEB31959, PRJEB31960	C57BL/6 female mice (32) CD-1 female mice (22)	-	No fibre diets (5 days prior to infection) Probiotics: Lactobacillus (9 species), Bifidobacterium (4 species), Streptococcus thermophilus, acacia gum, larch gum, galacto-oligosaccharide, L-glutamine, vitamin D3 (1 day prior to infection) Prebiotics (5 days prior to infection)	Dexamethasone 21-phosphate disodium (5 days prior to infection) Vancomycin and streptomycin (6 days prior to infection) Metronidazole (6 days prior to infection)	Mice given prebiotics or fibre diet shed fewer oocysts than those with no fibre diets. Increased oocyst shedding/severity of disease in mice with a reduced microbiome
2	VanDussen et al [40]	2020	E-MTAB-9100	HCT-8 Cells	-	Top 20 neonatal ICR mice metabolites (24 hours)	None	Capric acid, Monomyristin and Lauric acid reduced oocyst shedding. DHA, LnA and LA increased C. parvum prevalence
2	Karpe et al [41]	2021	-	3 week old, female, C57BL/6J mice (20)	-	No Intervention	None	Increase in short chain fatty acid synthesis, as well as D-amino acid retention in the gut. Increase in yeast proteins detected. Oxalate was accumulated in the liver of infected mice.

Figure 2. Shannon index scores (H) from studies described in Table 2. Shannon Index (H) values obtained from studies listed in Table 2. Order of the bars follows the order of Table 2 (descending) from left to right. Where specific Shannon Diversity Indexes could not be found within the study, the value was estimated from graphs provided within studies or the supplementary material. Mice studies indicated by mouse image, primate study highlighted with primate image, goat subjects indicated using a goat image. Images obtained from BioRender, then edited to remove colour.

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