Trichinella surveillance program in wild birds, Emilia-Romagna (northern Italy), 2006-2021. First report of Trichinella pseudospiralis in western marsh harrier (Circus aeruginosus) in Italy.

The nematode Trichinella pseudospiralis is a cosmopolitan parasite capable of infecting both birds and mammals including humans. T. pseudospiralis has a limited zoonotic importance in comparison to that of the other Trichinella species. However, it has been recognized as the etiological agent of two outbreaks of trichinellosis due to the consumption of wild boar meat. The role played by birds in the epidemiology of T. pseudospiralis is still unclear and needs to be deepened. The aim of our work was to show the results of an extensive wild bird surveillance carried out in the Emilia-Romagna (E-R) region, northern Italy, over the last 16 years. As part of the regional wildlife surveillance program, 14,933 raptors and carrion-eating birds' carcasses were necropsied from 2006 to 2021 and tested for the presence of Trichinella spp. larvae with only one positive result, an adult female of western marsh harrier. The larvae load (LPG) was evaluated on breast (100 LPG), wings (3.6 LPG), shoulder (2 LPG), head (4.5 LPG), thighs (8 LPG), lower legs (2 LPG) and tongue (0.77 LPG). The results of the present study confirm that also in northern Italy T. pseudospiralis has a low prevalence comparing to that of other Trichinella species. However, this study demonstrates that T. pseudospiralis can reach a high parasitic load in infested birds. The large distribution range, probably facilitated by its ability to infest birds, suggests the need for a continuous monitoring program.

Introduction

Nematodes of the genus Trichinella are relevant food-borne zoonotic agents causing trichinellosis in humans and show a global distribution in domestic and wild animals (Pozio and Murrell, 2006; Pozio, 2007). In Europe, four species with different biological characteristics, namely T. spiralis, T. nativa, T. britovi and T. pseudospiralis, are known to circulate in wild carnivores and some in sylvatic and domestic swine (Pozio, 2019).

The non-encapsulated species T. pseudospiralis, discovered for the first time in a raccoon (Procyon lotor) in Russia (Garkavi, 1972), is considered a cosmopolitan parasite (Pozio, 2016). This species is the only one of the genus capable of infecting both birds and mammals including humans (Pozio and Zarlenga, 2013; Pozio, 2016).

In the last years, there has been a great increase of the detection of T. pseudospiralis in the European Union. This nematode is widely distributed in wildlife (e.g. birds of prey, wild boars, red foxes, raccoon dogs), and its occurrence in domestic pigs and synanthropic rats was also documented (Pozio, 2001; Hurníková et al., 2005; Nöckler et al., 2006; Pozio, 2016; Cybulska et al., 2018), representing a potential source of human infections. The increased use of artificial digestion of muscle samples to detect Trichinella spp. larvae (European Commission, 2015), which is much more sensitive than trichinelloscopy in detecting non-encapsulated species (Gottstein et al., 2009; Bilska-Zając et al., 2020), is probably the primary reason for the increased recognition of this parasite (La Rosa et al., 2001). However, its prevalence is generally much lower than that of encapsulated sympatric species (Pozio and Zarlenga, 2013).

Therefore, T. pseudospiralis has a limited zoonotic importance in comparison to that of the other Trichinella species. However, it has been recognized as the etiological agent of two outbreaks of trichinellosis due to the consumption of wild boar meat, which occurred in France and Italy in 1999 and 2015, respectively (Ranque et al., 2000; Gómez-Morales et al., 2021).

The number of mammals tested for Trichinella spp. by digestion has been much higher than that of birds, thus the role played by birds in the epidemiology of T. pseudospiralis is still unclear and needs to be deepened (Pozio, 2016).

The aim of our work was to show the results of an extensive wild bird surveillance carried out in the Emilia-Romagna (E-R) region, northern Italy, over the last 16 years.

Material and methods

Sampling

According to the Commission Regulation (EC) no. 2075/2005 (European Commission, 2005), a wildlife monitoring program in the E-R region has been implemented in 2006 with the aim to provide data for the risk assessment of Trichinella spp. presence in domestic pigs.

The program has been carried out by the official veterinary services in collaboration with hunters, forest guards and staff employed in Wildlife Rescue Centres and identifies as indicator species of Trichinella presence all carnivores, birds of prey, and other bird species with necrophagous behaviour, such as corvids. As a food safety measure, the detection of Trichinella spp. is also carried out in all hunted wild boars (Regione Emilia-Romagna, 2022).

Diagnostic procedures in birds

From each bird, 10 g of muscles from the head were tested for the presence of parasite larvae by the magnetic stirrer method for sample digestion according to the Commission Regulation (EC) no. 1375/2015 (European Commission, 2015). This diagnostic procedure is considered as the gold standard for the Trichinella spp. larvae detection and does not differ from the ISO 18743:2015 procedure (International Organization for Standardization, 2015). In case of a positive result, larvae were recovered, stored in 90% ethyl alcohol and forwarded to the European Union Reference Laboratory for Parasites (EURLP, Istituto Superiore di Sanità, Rome, Italy) for species identification.

Trichinella species identification and isolate typing

DNA was extracted from single larvae using DNA IQ System kit (Promega, USA) and Tissue and Hair Extraction kit (Promega, USA). Five primer sets, targeting specific regions (expansion segment V, ITS1 and ITS2) of the ribosomal DNA repeats, were used in a multiplex PCR to obtain a species-specific electrophoretic DNA banding pattern (Zarlenga et al. 1999; Pozio and La Rosa 2010). PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and sent to Eurofins Scientific (Brussels, Luxembourg) for standard Sanger sequencing.

The PCR-amplified ESV sequence was compared to GenBank database, by Basic Local Alignment Search Tool (BLAST), to search for similar sequences. To investigate the polymorphism of ESV microsatellite locus, the sequence was aligned with published sequences of T. pseudospiralis isolates belonging to Palearctic, Nearctic, and Australian populations (Fig. 1). A total of six Palearctic isolates (northern Italy, southern Italy, Finland, Kazakhstan, southern Russia, Slovak Republic), one Nearctic isolate (Alabama), and one Australian isolate (Tasmania) were used in the alignment. All the analyses were done by using the bioinformatics platform CLC Workbench 8.0.1 (Qiagen, Hilden, Germany).

Fig. 1

Alignment of homologous ESV sequences of T. pseudospiralis isolates belonging to Palearctic, Nearctic and Australian populations. C. aeruginosus, isolate from the western marsh harrier (ISS8343); IT_rk, isolate from a red kite (Milvus milvus) of the Basilicata region (ISS7768); IT_wb, isolate from a wild boar hunted in Northern Italy (ISS2851); FI, isolate from a raccoon dog (Nyctereutes procyonoides) of Finland (ISS681); SK, isolate from a peregrine falcon (Falcus peregrinus) of the Slovak Republic (MN963194); RU, isolate from a raccoon (Procyon lotor) of Southern Russia (ISS13); KZ, isolate from a tawny eagle (Aquila rapax) of Kazakhstan (ISS176); US, isolate from a black vulture (Coragypus atratus) of USA (ISS470); AU, isolate from a tiger cat (Dasyurus maculatus) of Australia (ISS141). Conserved bases are represented by dots; gaps are represented by dashes; different residues are highlighted in red; TGC microsatellite region is boxed in green. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Results

As part of the wildlife monitoring program in the E-R region, northern Italy, 14,933 raptors and carrion-eating birds’ carcasses were necropsied from 2006 to 2021 and tested for the presence of Trichinella spp. larvae with one positive result (Table 1).

Table 1

Birds tested for Trichinella spp. in the Emilia-Romagna region, 2006–2021.

Order	Common name	Scientific name	No. of tested birds	No. of Positive
Accipitriformes	Northern goshawk	Accipiter gentilis	15
	Eurasian sparrowhawk	Accipiter nisus	114
	Golden eagle	Aquila chrysaetos	1
	Buteo	Buteo buteo	212
	Western marsh harrier	Circus aeruginosus	9	1
	Hen harrier	Circus cyaneus	1
	Greater spotted eagle	Clanga clanga	1
	Red kite	Milvus milvus	1
	European honey buzzard	Pernis apivorus	1
Charadriiformes	European herring gull	Larus argentatus	32
	Mediterranean gull	Larus melanocephalus	1
	Black-headed gull	Larus ridibundus	56
Falconiformes	Peregrine falcon	Falco peregrinus	1
	Hawk	Falco spp.	32
	Eurasian hobby	Falco subbuteo	7
	Common kestrel	Falco tinnunculus	346
	Red-footed falcon	Falco vespertinus	1
Passeriformes	Hooded crow	Corvus cornix	3829
	Eurasian jackdaw	Corvus monedula	7
	Crow	Corvus spp.	60
	Eurasian jay	Garrulus glandarius	1369
	Eurasian magpie	Pica pica	8324
Pelecaniformes	Great egret	Ardea alba	1
	Grey heron	Ardea cinerea	23
	Purple heron	Ardea purpurea	1
	Cattle egret	Bubulcus ibis	3
	Little egret	Egretta garzetta	8
	Least bittern	Ixobrychus exilis	1
	Dwarf bittern	Ixobrychus sturmii	1
	Black-crowned night heron	Nycticorax nycticorax	2
Strigiformes	Short-eared owl	Asio flammeus	1
	Long-eared owl	Asio otus	108
	Little owl	Athene noctua	210
	Eurasian scops owl	Otus scops	73
	Tawny owl	Strix aluco	39
	Barn owl	Tyto alba	42

On the 24th of March 2021, an adult female western marsh harrier (Circus aeruginosus Linnaeus, 1758), dead after an attempted rescue by a Wildlife Rescue Centre, was collected in the Modena municipality, E-R region, Italy; 34 m above sea level.

After the detection of Trichinella sp. larvae in head muscles (e.g. masseter and neck muscles), samples from other muscle groups as breast (5 gr), thighs (5 g), lower legs (5 g), wings (5 g), and the whole tongue (1.3 g) were also collected and tested for the presence of Trichinella sp. larvae. Following digestion, larvae were recovered, enumerated and the larvae per g of tissue (LPG) value was calculated. All the body parts tested positive for the presence of Trichinella sp. larvae with LPG values ranging from a minimum of 0.77 for the tongue to a maximum of 100 for breast muscles (Table 2). The larvae were identified as T. pseudospiralis by multiplex PCR. The amplified ESV sequence (GenBank accession No. OP325399) was identical to the homologous sequence obtained from T. pseudospiralis larvae isolated from a wild boar in Italy as well as to those isolated from a raccoon dog in Finland and from a peregrine falcon in Slovak Republic. When compared with other Palearctic isolates, including one collected from a red kite in Southern Italy, the sequence showed to differ only for the number of repeats of the microsatellite TGC (Fig. 1).

Table 2

T. pseudospiralis larvae distribution in the tested muscle groups of the western marsh harrier of this study. LPG = larvae per gram of tissue digested.

Muscle group	Total number of larvae	LPG
Breast	500	100
Wings	18	3.6
Shoulder	10	2
Head (masseter and neck muscles)	45	4.5
Thighs	40	8
Lower legs	10	2
Tongue	1	0.77

Discussion

Since its first description in a raccoon (Garkavi, 1972), T. pseudospiralis was detected in a large variety of domestic animals and wildlife including pigs, birds, wild boars, red foxes, racoon dogs (Pozio, 2016). Recently, the nematode was detected for the first time in a wolf from Italy (Ricchiuti et al., 2021). Birds are suitable host for this non-encapsulated species and are suspected to be involved in the maintenance and dissemination of the parasite in the sylvatic cycle. However, there have been more reports of natural infections of T. pseudospiralis in mammalian than in avian hosts, although these values may be biased given that wild birds are rarely tested for the presence of Trichinella spp. larvae (Pozio, 2016).

The reproductive capacity index values (RCI) of T. pseudospiralis isolates in experimentally infected chickens were apparently low (La Rosa et al., 2001), while rodents were more suitable hosts for the parasite, suggesting that the role played by mammals as reservoirs of this species is greater than that of avian hosts. Anyhow, this does not preclude the possibility that carrion-eating avian species may function as better hosts for T. pseudospiralis, resulting in a higher RCI, and therefore, enhancing the geographical dissemination of the species, which is known to be present in at least three continents (Pozio and Murrell, 2006).

The role of raptorial birds as a host of T. pseudospiralis has been documented in eight species belonging to five Families, namely: i) Family Strigidae: a tawny owl (Strix aluco) and a little owl (Athene noctua); ii) Family Corvidae: a crow (Corvus frugilegus); iii) Family Accipitridae: a tawny eagle (Aquila rapax), a western marsh harrier (Circus aeruginosus) and a red kite (Milvus milvus); iv) Family Tytonidae: a masked owl (Tyto novaehollandiae); v) Family Cathartidae: a black vulture (Coragyps atratus) (Pozio, 2016; Marucci et al., 2021).

A previous study on a larger number of scavenging birds evaluated the presence of Trichinella spp. in free-ranging corvids in central Italy. None of the 769 tested birds was positive (Mancianti et al., 2020).

Over a period of 16 years, the monitoring program of the E-R region involved several thousand birds belonging to six different orders and found only one positive result. In the same period and region, T. pseudospiralis larvae were also detected in only one out of thousands of screened wild boars (Merialdi et al., 2011), confirming the fact that the detection of this parasite is sporadic (Pozio, 2016).

The T. pseudospiralis-infected subject identified in our study was rescued by a Wildlife Rescue Centre because it was unable to fly. The routine necropsy showed a wing fracture and cranial trauma as the only pathological findings. After the positive result, the subject was more thoroughly examined to determine the distribution and load of T. pseudospiralis larvae among different muscular groups. The higher LPG (value 100) was detected in the breast muscles, while the average number of larvae in other muscular districts was significantly lower (Table 2).

The distribution of muscular larvae of Trichinella spp. is mainly influenced by both the species of nematode and the host species (Kapel et al., 1998; Cybulska et al., 2018). According to Kapel et al. (2005), the predilection muscles of T. pseudospiralis larvae in experimentally infected red foxes were the diaphragm, the upper forelimb and the filet, whereas a lower larval recovery was observed in the lower hindlimb, the masseter, the lower forelimb and the tongue. On the contrary, in an experimentally infection in swine, the highest larval burden was detected in the tongue and the diaphragm (Pozio et al., 2020).

The intensity of Trichinella spp. in wild animals is generally lower than 10 LPG, while intensities higher than 50 LPG are considered exceptional (Dick and Pozio, 2001). Interestingly, a previous report on T. pseudospiralis in Circus aeruginosus from Tasmania showed a parasite load of 650 LPG. In the same report, a 6-day-old marsh harrier was experimentally infected with muscle tissue containing T. pseudospiralis larvae (34 LPG) derived from a naturally infected Tasmanian devil, highlighting that this bird was a suitable host for T. pseudospiralis (Obendorf and Clarke, 1992). To the best of our knowledge there are no studies on pathological changes in birds infected by Trichinella larvae. Therefore, we are not able to establish whether the observed fractures were caused by injuries due to a pre-existing pathological flying difficulty linked to the high larval burden in breast muscles.

The western marsh harrier is a medium-sized diurnal raptor belonging to the Family Accipitridae Vieillot, 1816. The marsh harrier diet consists mainly of small mammals such as mice (Apodemus spp.) and voles (Arvicola spp.), and fish, insects, eggs and medium-sized birds (Alves et al., 2014). This feeding behaviour makes this bird a susceptible species for Trichinella spp. infection. The species has an extremely wide breeding range from Europe and north western Africa to Central Asia and the northern parts of the Middle East. The species is a partial migrant, with populations in Southern and Western Europe, North Africa and at the south of its range in Asia being generally resident (Ferguson-Lees and Christie 2001; Agostini and Panuccio, 2010). In Italy, breeding sites are mainly located in coastal wetlands of the northern Adriatic and inland marshes of the Po Valley (Nardo, 1993; Martelli and Rigacci, 2005; Spina and Volponi, 2008).

Biological, biochemical, and molecular analyses have revealed three different populations of T. pseudospiralis in the Palearctic, Nearctic and Australian regions (Zarlenga et al. 1996; La Rosa et al., 2001). The ESV sequence of the larvae infecting the western marsh harrier of this study showed to be in some cases identical, in others highly similar, to the homologous sequence obtained from T. pseudospiralis larvae isolated from different hosts in Europe. The difference concerned only the numbers of repeat of the TGC microsatellite. Since the TGC microsatellite length polymorphism represents a peculiar characteristic of the Palearctic isolates of T. pseudospiralis (La Rosa et al., 2001), the high variability of the region can explain the difference between the sequences obtained from the larvae belonging to the two Italian birds of prey.

Conclusions

The results of the wildlife monitoring program of the Emilia-Romagna region confirm that also in Italy T. pseudospiralis has a low prevalence comparing to that of other Trichinella species (e.g. T. britovi), especially in carnivores (Garbarino et al., 2017; Ricchiuti et al., 2021). However, this study demonstrates that T. pseudospiralis can reach a high parasitic load in infested birds. Although the low prevalence of this species represents a limited zoonotic risk for humans, the sporadic but at the same time ongoing isolation of this parasite suggests a stable presence in the Italian territory. The large distribution range, probably facilitated by its ability to infest birds, suggests the need for a continuous monitoring program involving both domestic and wild animals.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.