Toxocara cati and Toxoplasma gondii in French Birds of Prey

Previous studies have described that 75% of zoonotic pathogens, including some nematodes and protozoa, have a wild reservoir and that 65% would rely on a community of reservoir hosts rather than a single reservoir host. Interestingly, bird species are among the 38 reservoir hosts of pathogens ranked as priority species to watch (Plourde et al. 2017). Raptors (and some other bird species) eat many small mammals or scavenge carcasses that may host meat-borne parasites. Such species may therefore be helpful to monitor the circulation of these parasites. Because most of these birds are protected species, and many potential study methods are considered too invasive, their pathogens are under-reported. Raptors can host many apicomplexan parasites, such as Toxoplasma gondii (Mancianti et al. 2020), Sarcocystis spp., (Dubey et al. 1991), and Neospora caninum (Gazzonis et al. 2021), and nematodes, being definitive hosts for Trichinella pseudospiralis (Pozio 2016) and paratenic hosts for Toxocara spp. (Marucci et al. 2013).

We assessed the potential use of bird muscles for the direct detection of nematodes as well as improving their detection from this tissue. Additionally, we tested the use of muscle fluids for detecting the presence of antibodies against T. gondii.

Sampling was approved by the Scientific Council of Normandy's natural heritage and by the Regional Directorate for the Environment, Planning and Housing of Occitania and was carried out in accordance with the relevant national legislation. These samples were taken from recently dead birds at three wildlife rescue centers: Hôpital de la faune sauvage des Garrigues et Cévennes, Ganges (Hérault) located in middle South of France; Centre de preservation de la faune sauvage C.H.E.N.E., Yvetot (Normandie) located in North of France; and Hôpital de la faune sauvage de l'Ecole Nationale Vétérinaire d'Alfort (Chuv-FS EnvA), located in the Paris region (Ile-de-France). At least 5 g of the pectoral muscles were collected from 22 different bird species during 2017 and 2018 (Table 1). After being collected, the muscles were frozen at –20 C until they were processed.

After thawing, samples were pooled according to year of collection, geographic origin, and bird species (Table 1). Nematodes were isolated from muscle pools using HCl-pepsin digestion (Gamble et al. 2000). The second year of the study, a second, 30-min decantation step was added to allow more time for lighter larvae to sediment. If the conservation of the morphologic criteria allowed it, an attempt to identify nematodes to genus level was carried out (Karadjian et al. 2020).

We performed DNA extraction on single larvae following the protocol for Trichinella larvae identification (Zarlenga et al. 1999; Karadjian et al. 2017). A first step for identification based on molecular biology was performed by amplifying a pan-nematode 18S fragment when the morphology did not allow genus identification; then, a fragment including a part of 5.8S ribosomal gene and the complete internal transcribed spacer 2 (ITS2) region was amplified (Karadjian et al. 2020). Positive reactions were followed either by sequencing, the first year, or to develop a faster internal method the second year by restriction fragment length polymorphism (RFLP). This later was performed at 37 C for 2 h in 15 µL of mix containing 6.8 µL of RNase/DNase free water, 1 µL of CutSmart® Buffer, 2 units of AscI restriction enzyme, 4 units of BsiWI-HF restriction enzyme, 2 units of CspCI restriction enzyme, 20 µM of Sadenosylmethionine to activate CspCI (all components NEB, Evry, France), and 6 µL of the ITS2 PCR products. On 1.5% agarose gels, the Toxocara cati species profile consists of 100 base pair (bp), 160 bp, and 260-bp bands while the Toxocara canis profile consists of 150 bp, 310 bp, and 360-bp bands (Fig. 1).

Fluids from bird muscles were obtained after thawing at room temperature. Immediately after being defrosted, they were analyzed by the modified agglutination test (Dubey and Desmonts 1987) for the detection of Toxoplasma gondii-specific antibodies using an antigen prepared from formalin-fixed whole RH3 tachyzoites (Villena et al. 2012). The starting dilution was 1:6. Seven further twofold dilutions were made, up to 1:768. To score animals positive or negative, we used the cut-off value 1:6.

We recovered 277 nematode larvae from bird muscles following digestion (Table 1). A total of 33/76 (43%) muscle pools collected over the 2 yr at the three different sites were positive. In Normandy, the Cevennes, and Ilede-France, respectively, 11/26 (39%), 13/33 (39%), and 9/15 (60%) pools were positive. From the 2017 cohort, 11/33 (33%) of the pools were positive, including 5/12 (42%), 1/ 12 (8%), and 5/9 (56%) from Normandy, the Cevennes, and Ile-de-France, respectively. For the 2018 cohort, 22/43 (51%) of the sample pools were positive, including 6/16 (38%), 12/21 (57%), and 4/6 (67%) from Normandy, Cevennes, and Ile-de-France, respectively.

The larvae of 19/33 positive pools could be assigned to the genus Toxocara (Table 2). Some larvae were too damaged to allow any visual identification. Nevertheless, they had a pointed posterior part and a rounded anterior part like all larvae of the genus Toxocara (Fig. 2), and the larval length (350–400 µm) and width (18–22 µm) were the expected sizes for larvae of the genus Toxocara (Euzéby et al. 2005). The PCR 18S confirmed this: PCR ITS2 products from larvae of the first-year cohort were sequenced (Table 2) whereas a RFLP was performed for larvae of the second-year cohort; they all belonged to the Toxocara cati species (Table 2).

Muscle fluids were recovered for 166 samples from 18 bird species. We detected antibodies in 65 samples of 11 species (Table 3). Antibody titers ranged from 1:6 to 1:384, as follows: 34, 18, eight, three, and one samples were positive at 1:6, 1:12, 1:24, 1:48, 1:96, and 1:384, respectively. The overall percentage of Toxoplasma gondii infection in birds was 39% (65/166) with relatively close percentages for the three sampled regions, Cevennes, Ile de France, and Normandy: 32% (8/25), 35% (8/ 23), and 42% (49/118), respectively.

No species-specific pattern was detected for Toxoplasma gondii percentages, with positive results ranging from 25% (1/4) to 100% (1/1). When looking at species with >10 samples, all were 25–51%: 25% (5/20), 32% (6/19), 40% (12/30), 50% (9/18), and 51% (21/41) for Falco tinnunculus, Strix aluco, Tyto alba, Larus argentatus, and Buteo buteo, respectively.

We adapted a technique previously standardized for Trichinella larvae detection in muscle. It allows the release of nematodes, which may sometimes belong to species other than Trichinella spp., from the muscle tissue (Marucci et al. 2013, Karadjian et al. 2020). For the samples from 2018, we added a second, 30-min decantation to increase the chance of collecting smaller and slimmer larvae that would not have time to settle down during the first decantation. We found 19 pools to be positive on the first decantation and 13 pools were positive in the second decantation, with three of these having been negative on first decantation; nine of the pools that were positive on the first decantation did not release further larvae on the second decantation. Among the 10 pools positive in both decantations, eight had as many or more larvae after the second decantation than after the first. We found Toxocara cati in 10/17 raptor species studied, 15 of which feed on small mammals, especially rodents. Of the seven bird species that did not have Toxocara cati, two do not feed on small mammals (Circaetus gallicus and Pandion haliaetus). Of the 10 nonraptor bird species, only three had Toxocara cati (Larus marinus, L. argentatus, and Bubulcus ibis); small mammals are included in the diet of these species. Moreover, embryonated eggs released by cats on the environment could also infect birds, as it happens in all paratenic hosts. It is therefore likely that when birds share an environment and prey with cats, to a greater extent they have a higher risk of being infected with Toxocara cati.

For Toxoplasma gondii infection, detection was positive in 8/11 raptor species and only 3/ 7 nonraptor species; overall, 65/166 (39%) of samples tested positive. Our results are in concordance with those obtained in France by Aubert et al. (2008), who found an overall percentage of 50% based on sera samples. The differences in percentages could be a mirror of what happens in prey species, and probably birds with lower parasite yields come from areas populated by prey animals weakly parasitized. Thus, raptors could be playing a crucial sentinel role for monitoring infections by Toxoplasma gondii. An interesting finding is that B. buteo presented a constant percentage in the three regions studied, with a mean at 52%±8% (21/41), which is consistent with the 79% (11/14) that Aubert et al. (2008) found for the Nouvelle Aquitaine region. In contrast, we found 5/20 (25%) Falco tinnunculus positive while Aubert et al. (2008) found 0/8 (0%) positive. These data suggest that the sampling plan (number of samples, location, etc.) must be considered before making any conclusions about the role of a given species in Toxoplasma gondii transmission.

In conclusion, we confirmed antibodies against Toxoplasma gondii in muscle fluid and Toxocara cati in muscles of wild birds in France. Working with the network of wildlife rescue hospitals, raptors could be good sentinels to monitor the circulation of these two parasites among wild birds. These hospitals play a crucial role in the care of injured, diseased, and exhausted animals, and could become a cornerstone of wildlife diseases monitoring.