Understanding how parasitism may affect social behavior and social networks is key to understanding the impact of infection on a population. Infection can disrupt social networks by altering the behavior of both infected individuals (e.g. by reducing activity) and the behavior of uninfected individuals (e.g. avoiding sick individuals), both of which can have an impact on social group dynamics and parasite transmission. Here we test experimentally how parasitism affects social contact behavior and social network structure using a common parasite infection of sheep. Three treatment groups, each with 4 replicate social groups were established (i) Parasitised; all lambs were infected with a parasitic nematode, (ii) Non-parasitised; all lambs remained uninfected (iii) Mixed; part of each group were infected, and part of the group remained uninfected. Contact behaviours of each individual were recorded using proximity loggers during four phases of infection (pre-parasite, pre-patent, patent-parasite, post-parasite). We found infected individuals in the parasitised and mixed groups reduced contact frequency following infection. Infected individuals in mixed groups however reduced contact frequency to a greater extent than infected animals in the fully parasitised group. Despite the reduction in contacts between infected animals in the mixed group, the social network structure was unaffected, as non-infected individuals maintained pre-parasite levels of social interactions with their infected conspecifics. These results demonstrate how infection can impact the social behavior of all animals within a group, and how the expression of behavioral change may depend on the parasitic status of all group members and the response of uninfected conspecifics.

Animals and experimental design

Sixty 12-week-old Texel x Bluefaced Leicester lambs were selected randomly from a commercial flock of sheep that had been reared indoors since birth, under conditions that excluded nematode infection and so were considered parasite naïve. The lambs were divided into one of three treatments with 4 replicate groups of 5 lambs within each treatment. These were (i) Parasitised: all lambs were experimentally infected with parasitic nematode T. circumcincta and were of the same parasitic status, (ii) Non-parasitised: all lambs were dosed with water, remained parasite naïve, and were of the same parasitic status and (iii) Mixed: a group containing animals of mixed parasitic status, where three animals were dosed with water and two were experimentally infected with T. circumcincta larvae. Each replicate group was balanced for sex (three females and two males per group) and weight (live mean weight ± standard deviation 27.6 ± 0.13Kg). To choose the individuals within the mixed group to be infected, a structured approach was chosen whereby the smallest female and largest male in all groups were infected. This approach was chosen to account for any potential effect of sex and weight, and so reduce the residual variation, thus increasing the power to detect the effect of parasitism in these groups. To ensure all animals within each group had similar social experiences with conspecifics, no siblings were allocated to the same group. One week before the experiment start date, groups were put onto pasture in individual plots laid out in a six-by-two grid, with each plot measuring 30x30m and separated by sheep netting. All plots had been free from grazing ruminants for the previous three years and animals were given ad-lib access to water. Diet selection can impact sheep movement and thus social contact behaviour. Therefore, in order to minimise any effect of plot difference in vegetation availability, groups were rotated clockwise around the plots twice weekly, so each plot had animals from each treatment group for the same amount of time.

The experiment was conducted in summer 2019. The experimental timetable (a total of 9 weeks) was divided into four phases; pre-parasite (week 1), a period when all lambs would be kept parasite naïve; pre-patent (weeks 2–4), a period when lambs identified for infection would be parasitised but not yet showing any pathological or physiological effects of parasitism and are not yet shedding eggs; patent-parasite (weeks 5–7), a period when lambs show physiological responses to infection (e.g. reduction in weight gain and gut wall damage) and are shedding eggs in their faeces; post-parasite (weeks 8–9), a period after all lambs were dosed with anthelmintic, and considered parasite free. On the first day of week two, lambs identified for infection, which included all lambs in the parasitised groups and two out of five lambs in the mixed groups received an oral dose of 5,000 L3 stage T. circumcincta larvae, and lambs not identified for infection were handled in the same way and received a dose of water. All lambs were then trickle-dosed with either water or T. circumcincta larvae 3 times per week for 6 weeks. The trickle infection chosen (5,000 L3/day) would ensure a subclinical infection would be established and also represented an infection level similar to that encountered by sheep naturally when grazing on contaminated pastures (Coop et al. 1982; Wood et al. 1995). On the first day of week 8 all lambs were treated with anthelmintic (Albendazole, 1ml/10Kg) and infections were cleared. The experiment was designed to operate within the life cycle of the parasite so that there was no risk of natural parasite exposure of our experimentally infected individuals. At the end of the study, all lambs were returned to a commercial flock.

Animal measurements 

On the first day of each week, faecal samples were taken from all sixty lambs to check for presence/absence of faecal eggs and to estimate the number of nematode eggs per gram of faeces (epg) in positive samples using a modified salt-flotation method (Jackson, 1974). Blood samples were taken by jugular venepuncture at the start of weeks 1, 7 and 9 (one measurement during pre-parasite, patent-parasite and post-parasite phases respectively) to measure serum pepsinogen levels (an indication of parasite-induced abomasal damage) using a sheep pepsinogen ELISA assay kit (BlueGene Biotech, Shanghai, China). To assess the impact of infection, lambs were also weighed to measure weekly weight gain. At the end of the experiment, a faecal sample and weight measurement were taken from every animal to assess the lambs' final weights and parasite load.

After collection, faecal samples were weighed out into 1g subsamples and stored at 4°C for faecal egg counts. Blood samples were spun within two hours of collection at 3660 r.p.m at 4°C for 15 minutes, the serum was removed and stored at -20°C to measure serum pepsinogen levels.

Measuring contact behavior

Contacts between lambs were continuously recorded using proximity loggers (Sirtrack Ltd., Havelock North, New Zealand). Each lamb in the study was fitted with a proximity data logger on a neck collar to record close proximity contacts with any other individual in their social group. The proximity loggers use an ultra-high frequency (UHF) to send out signals to other loggers using a unique code, while receiving signals from other loggers. The detection distance was set to 1–1.5m, to allow detection of a close-contact situation, during which social interactions might occur (Ozella et al. 2020). Once a contact is detected by a logger, a contact is recorded until one of the loggers in the contact fails to receive a signal for longer than the separation time, which was set at 10 seconds. When two lambs came into contact the time, date, logger ID, recipient logger ID, and duration of the contact were recorded.

Any contacts that were recorded before loggers were placed on the lambs, while animals were being handled during the experiment, or occurred between animals in neighbouring plots were not included in the analysis. All contacts of 1 second or less were removed, as it is believed these may represent weak collar signals (Drewe et al. 2012), or detection signals at the edge of the detection range (Prange et al. 2006). To reduce inter-logger variation that has been associated with proximity loggers (Drewe et al. 2012; Boyland et al. 2013), loggers were rotated between social groups twice weekly. As reciprocal contact data from two different collars are not completely symmetrical due to reflection, refraction and absorption of radio waves (Patison et al. 2010), the contact duration between two loggers was defined as starting when one logger recorded a contact and ending when either logger failed to maintain a contact (Hamede et al. 2009; Patison et al. 2010; Smith et al. 2019).