Trichinella britovi Larval Biomass in Wild Canids in the Abruzzi Region, Italy

: This study estimated the larval biomass of larvae of Trichinella britovi in wild canids in annual batches of carcasses recovered from the Abruzzi region, Italy, during 2013–2020. The larval biomass decreased from about six million larvae in 2013 to less than three million in 2019–2020. The province of L’Aquila (99 positive specimens out of 109) was the territory most interested by Trichinella infection. As the larval biomass correlates with the larvae per gram, muscle mass and number of positive carcasses, it could be a better indicator to assess Trichinella infection in wildlife than the prevalence alone, which correlates with only positive carcasses and the examined ones.


Introduction
Wild canids play an epidemiological role in maintaining sylvatic cycles of nematodes of the genus Trichinella that spread within these populations through carnivorism both via predator-prey and by their scavenger behavior. In European countries, Trichinella infection in wild canids is prevalently due to Trichinella britovi and T. nativa and, to a lesser extent, to T. spiralis and T. pseudospiralis. In particular, the wolf (Canis lupus), red fox (Vulpes vulpes) and raccoon dog (Nyctereutes procyonoides) are mainly reported as host-reservoirs in Eastern and Central Europe and in Central Balkan [1].
In Italy, except for single reports due to T. spiralis in red fox and T. pseudospiralis in wolf [1,2], the species found in wild canids is T. britovi [3,4].
Currently, the trend of Trichinella infection in wildlife is based on the apparent prevalence (AP), intended as the proportion of positive samples to total tested. More recently, studies on global Trichinella larval burden in positive specimens have pointed to estimate the biomass of parasites as a different way to understand the epidemiological patterns of Trichinella infection in wildlife [5,6].
The aim of this study is to update data on T. britovi infection in wildlife and to estimate the larval biomass of parasites in the annual batches of wolf and red fox carcasses recovered from territory of the Abruzzi region.

Animals, Area Studied
Abruzzi is a region of Central Italy with a surface of 10,831 km 2 and a population of about 1.3 million inhabitants; approximately 35% of its territory is occupied by national and regional parks or nature reserves. In the 2015-2020 period, as part of the regional plan on wildlife health monitoring, the carcasses of susceptible species recovered from regional territory (injured animals, illegally hunted, poisoned or diseased animals), were investigated for infective larvae of Trichinella using at least 5 grams of muscle taken from the main muscle predilection sites (diaphragm or lower forelimb or lower hind limb). In addition, data from official control of wild boars (Sus scrofa) for human consumption were considered in the investigation. Samples were analyzed by enzymatic digestion methods according to European Regulation 1375/2015 [7]. For each positive sample, the larvae were numbered as larvae per gram (LPG), stored in 96% ethanol and forwarded to the International Trichinella Reference Center (ITRC, Rome, Italy) for species attribution by multiplex PCR according to a published protocol [8].

Data Collection and Normalization, Larval Biomass Estimation
For each animal investigated in the considered period, the weight, sex and location of recovery were recorded as well as laboratory data (muscle site, LPG, species attributed to Trichinella). In addition, similar data from a previous study [4], limitedly to 2013 and 2014, were considered.
Among T. britovi positive specimens, seven wolves and five red foxes' carcasses were selected for a preliminary study on the larval distribution of T. britovi in these species, according to a boning protocol described by Kirjušina et al. [6]. The following parameters were derived from this study and used for data normalization: • Muscle coefficients, as ratio of average muscle mass weight to total weight; • LPG coefficients, as ratio of average LPG of single muscle site to global LPG • Lower and upper confidence interval (CI95%) of LPG coefficients.
Finally, the larval biomass of each positive specimen investigated during 2013-2020 period was estimated by the formula: The overall larval biomass in the annual batch of wolf and red fox carcasses was estimated by summing the respective individual larval biomass. Table 1 summarizes the results of the investigation on Trichinella infection in susceptible hosts in which at least one sample was tested positive. Overall, 109 strains were detected and typed as T. britovi, except 17 which were not typable.  Positive specimens came prevalently from the province of L'Aquila (99 out of 109), where the overall AP during the period was 1.3%, but it reached 22.8% in wild canids (wolf, 37.7%; red fox, 13.4%).  Positive specimens came prevalently from the province of L'Aquila (99 out of 109), where the overall AP during the period was 1.3%, but it reached 22.8% in wild canids (wolf, 37.7%; red fox, 13.4%).

Coefficients Used for Data Normalization
The following parameters were derived from the study on T. britovi larval distribution in seven naturally infected wolf and five red fox carcasses:   In general, the level of dispersion of the observed data was high. In fact, the difference between the average upper and lower confidence intervals was higher than the respective averages of observed values of AP and larval biomass (data not shown). In general, the level of dispersion of the observed data was high. In fact, the difference between the average upper and lower confidence intervals was higher than the respective averages of observed values of AP and larval biomass (data not shown).

Discussion
Our investigation has certainly had limits relating to the lack or the low representation of certain host species/families of Trichinella, as well as the measure uncertainty in the estimations. However, samples coming from 296 out of the 305 municipalities of the region (data not shown) adequately covered the studied area which was investigated for a sufficiently long number of years. Furthermore, our methodology on estimation of Trichinella larval biomass has been adapted for the first time to the wolf and red fox.
The study on AP in 2015-2020 has updated a previous investigation related to the 2004-2014 period [4], confirming a swinging trend around an average of 14.4% of the infection in wilds canids, slightly increasing from the previous observation, and the sporadicity of infection in wild boar and Mustelidae. The L'Aquila province, where the Abruzzi, Molise and Lazio National Park, the Gran Sasso and Laga Mounts National Park and the Sirente-Velino Regional Park are prevalently located, continues to be by far the territory more interested by the Trichinella infection with more than 90% of positive specimens.
The study on T. britovi larval biomass in 2013-2020 has emphasized the role of the wolf as a reservoir host of the parasite, and further minimized the role of the red fox, thanks to its greater muscle mass which could be infected (Figure 1). The larval biomass trend, contrary to the prevalence trend, has decreased in the period of observation from about six million larvae to less than three million in 2019-2020. Moreover, this trend with high peak and strong decreases has strengthened our hypothesis that this was due to the effect of removing the agent from the environment through recovering carcasses and it can be considered a measure of this good practice ruled by the regional wildlife monitoring plan.
Finally, as the larval biomass trend correlates with the larvae per gram, muscle mass and positive carcasses, it could be a better indicator to assess Trichinella infection in wildlife than the apparent prevalence alone, which correlates with only positive carcasses and the examined ones.