Data for: Co-infection, but not infection intensity, increases shedding in a gastrointestinal helminth of gamebirds

Description

Notes

Methods

Study system and sample collection

Ring-necked pheasants (Phasianus colchicus) were collected postmortem between November and December 2023 from 8 recreational pheasant shoots across south-west England. Pheasants were sexed and body size (beak - cloaca length, to the nearest 0.5cm) and body mass (to the nearest 0.5g) were recorded. Scaled mass index (SMI) was calculated as a measure of body condition following (Peig and Green, 2009). We calculated SMI separately for males and females to account for sexual size dimorphism.

A faecal sample was collected directly from the cloaca and refrigerated at 4°C within 10 hours of collection. The lower digestive tract (small intestine, large intestine, and caeca) was removed and immediately stored in 10% formalin. In total, faecal samples and intestines from 58 pheasants were obtained.

This research was conducted under the approval of the Ethical Committee of the University of Exeter (ethical approval number 513904). The study was conducted in accordance with all relevant ethical regulations and principles. All pheasants were released under license and killed under the Game Act 1831.

Quantification of shedding intensity

A modified McMaster method was used to quantify shedding intensity (Levecke et al., 2011). 0.5g of faecal matter was suspended in 7ml of sodium nitrate flotation solution (specific gravity 1.2 +- 0.05) (VetLab Supplies). The suspended sample was homogenised and strained to remove any large debris. 0.5ml aliquots were added to two slide chambers on a McMaster slide. Slides were visually examined using light microscopy at 40x magnification. The number of *H. gallinarum *eggs on each slide was recorded. Slides were also examined for evidence of secondary helminth infections and the presence of Eimeria spp. oocytes. Eggs of Syngamus trachea and *Capillaria spp. *are challenging to visually distinguish, so were recorded only the presence or absence of co-infecting helminths. Published keys assisted in the morphological identification of helminth eggs and Eimeria spp. oocytes (Deviyanti et al., 2023, Goldová et al., 2006, Metwally et al., 2020). Counts were expressed as eggs per gram (EPG) and oocytes per gram (OPG), for helminths and Eimeria spp., respectively. This was obtained by multiplying the sum of both chambers by 50.

Quantification of infection intensity

Intensity of infection was quantified as the number of helminths present in the lower digestive tract. For each sample, the digestive tract was opened longitudinally and flushed with running water over a fine mesh sieve (aperture of 150 mic). Helminths collected in the sieve were retained, identified (Tanveer et al., 2015) and counted. The lining of the digestive tract was also examined. *H. gallinarum and Capillaria spp. *were observed in the sampled pheasants. Infection intensity quantification was conducted blind with respect to faecal egg counts.

Statistical analyses

We used a generalised linear model with a negative binomial error structure to identify predictors of *H. gallinarum shedding. A *negative binomial error structure was used to account for overdispersion. *H. gallinarum *eggs per gram (EPG) of faeces was included as the response variable and counts of *H. gallinarum *adults found in the digestive tract, host sex, host body condition, sampling location and co-infection status (co-infection / no co-infection) were included as explanatory variables. The duration of faecal sample storage was included as an additional covariate to account for possible sample degradation over time (Crawley et al., 2016).

Second, we explored the role of co-infection on *H. gallinarum *shedding in more detail using generalised linear models with a negative binomial error structure. All models included the same variables as the initial model but varied in their measure of co-infection: a) co-infection with another helminth (yes / no), b) co-infection with Eimeria spp. (yes / no), and c) the number of detected co-infections (none, one, two).

Finally, we used a generalized linear model with a negative binomial error structure to identify predictors of H. gallinarum infection intensity.* *Counts of *H. gallinarum *in the digestive tract were included as the response variable and host sex, host body condition, sampling location, and co-infection status were included as explanatory variables.

All models were fitted using an ordinary least squares framework and inspected for homogeneity of variance, normality of error structures, linearity and overdispersion. Significance of factors was obtained by comparing two nested models, with or without variables of interest, using likelihood ratio tests. Host body condition and H. gallinarum infection intensity were scaled to aid model conversion.

All analyses were conducted in R version 4.3.0 (R Core Team, 2013) using the packages lme4 (Douglas Bates et al., 2015), tidyverse (Wickham et al., 2019), , DHARMa (Hartig, 2018), ggplot2 (Wickham, 2016), MASS (Venables and Ripley, 2013), patchwork (Pedersen, 2019), and gg.gap (Jiacheng Lou, 2019).

References

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