A. Humski, M. Mitak, M. Mikulić, D. Stojević, B. Šimpraga, F. Krstulović, B. Ječmenica, T. Amšel Zelenika and L. Jurinović*
Gulls, as migratory wild birds are known to spread different pathogens over long distances. The aim of this study was to access the prevalence of different Listeria species in a gull population feeding at a landfill in Zagreb. In total, 390 gulls of three species were sampled: Yellow-legged Gull, Larus michahellis; Black-headed Gull, L. ridibundus and Common Gull, L. canus. The most prevalent species was Listeria innocua (14.4%), while L. monocytogenes was found in 11.3% of tested samples. Other species were present in a smaller percentage; L. welshimeri (1.3%), L. ivanovii (0.5%) and L. seeligeri (0.3%). Serotyping of L. monocytogenes isolates was performed using molecular and conventional methods, and most isolates belonged to the 1/2a and 1/2b serotypes. To the best of our knowledge, this is the first report of the presence of L. monocytogenes and other Listeria spp. in wild birds in Croatia.
Key words: wild birds; zoonosis; Listeria monocytogenes
The genus Listeria includes several species that are closely related both morphologically and biochemically. The majority of these Gram-positive, rodshaped, facultative aerobic-anaerobic, non-spore forming, psychotropic and ubiquitous bacterial microorganisms are non-pathogenic. Only two members of this genus are considered pathogens, Listeria monocytogenes and Listeria ivanovii that cause infection in humans and animals, respectively (Seeliger and Jones, 1986; Guillet et al., 2010; Beye et al., 2016).
In view of public health, listeriosis caused by Listeria monocytogenes is of undisputed importance. This zoonosis is primarily reported as a food-borne disease. There are many paths by which L. monocytogenes enters into the food chain, as this species is omnipresent. However, infection is mainly linked with the consumption of contaminated ready-to-eat foods whose physicochemical characteristics allow the survival and growth of this pathogen to quantities sufficient to cause disease (EFSA and ECDC, 2021).
Similar to other opportunistic infections, the occurrence, clinical symptoms and the outcome of listeriosis depends on the status of the host’s immune system, virulence characteristics of the present bacterial strain and the ingested dose.
While most people ingest it relatively frequently in small amounts and do not develop symptoms, individuals immunocompromised for any reason are prone to develop listeriosis (Swaminathan and Gerner-Smidt, 2007; Orsi et al., 2011).
Two forms of listeriosis are significant: gastroenteritis, and invasive infection.
Though this is a relatively rare disease, it is characterized by a high mortality rate, and therefore is placed at the top of the list of food-borne diseases with a fatal outcome (EFSA and ECDC, 2021).
A number of discriminatory subtyping methods of L. monocytogenes strains (PFGE; MLST; WGS) have been introduced for the purpose of epidemiological analyses, while serotyping remains significant as the first step of initial classification. Based on the combination of somatic and flagellar antigens, L. monocytogenes isolates are categorised into 13 different serotypes (Seeliger and Jones, 1986; Farber and Peterkin, 1991; Graves et al., 1999) and further distributed into at least four phylogenetic lineages (I, II, III, and IV) that are differentiated by the nucleotide sequences of three genes (flaA, iap, and hly) (Nightingale, 2010; Orsi et al., 2011; Zhang et al., 2016; Wang et al., 2019).
While L. monocytogenes lineage II strains (serotype 1/2a) are more widespread in the environment and in foods, and cause listeriosis in animals and sporadically in humans, lineage I strains (serotypes 1/2b and 4b) are responsible for most of the invasive listeriosis outbreaks worldwide (Wiedmann et al., 1996; Orsi et al., 2011).
Listeria reservoirs include a variety of wild and domestic birds and other animals, in both the natural and the urban environment (Sotirios et al., 2007; Vivant et al., 2013). It is well documented how wild and migratory birds represent long-distance vectors for different types of microorganisms that can pose a risk to humans (Nuttall, 1997; Kapperud et al., 1998; Thornley et al., 2003; Ejidokun et al., 2006). However, the involvement of free-living birds in supporting reservoirs and in the transmission of L. monocytogenes to humans has not yet been fully elucidated (Hellström et al., 2007).
The objective of this study was to investigate the presence of L. monocytogenes and other Listeria species in gulls as one of the most common birds living and feeding in the urban environment through species identification and the detection of serotypes, in order to determine their prevalence and possible zoonotic potential as a risk to human health.
Materials and Methods
Sample collection, bacterial isolation and identification
During the winter months (November, December, January) of three consecutive years (2018, 2019, 2020) a total of 390 cloacal swabs were taken from the gulls captured using a cannon net at a landfill site in Zagreb (45.45 N, 16.01 E). Gulls belonged to three species (Yellow-legged Gull, Larus michahellis; Black-headed Gull, L. ridibundus and Common Gull, L. canus), with Black-headed Gull as most abundant.
The detection and isolation of Listeria spp. was performed according to the method EN ISO 11290-1:2017.
Immediately after swabbing, collected cloacal swabs were placed in tubes with half-strength Fraser broth (primary enrichment) (Merck), and transported at ambient temperature to the laboratory to start incubation within eight hours from sampling. After incubation at 30°C for 25 h ± 1 h, a loopful (10 µL) of the primary enrichment cultures were streaked on the surface of two selective plating mediums: Agar Listeria according to Ottaviani and Agosti (Oxoid), and Oxford agar (Bio-Rad). Further, 0.1 mL of the same broth cultures was added to tubes containing secondary enrichment medium (Fraser broth, Merck). The secondary enrichment medium was incubated for at 37°C 24 h ± 2 h, and the surface plating-out procedure was repeated on the same two selective media. Selective agars were incubated at 37°C for a total of 48 h ± 2 h.
All presumptive Listeria spp. colonies were isolated from each sample, and subcultured onto Tryptone Soya Yeast Extract Agar (TSYEA) plates (TSA; Oxoid/YE [0.6%]; Oxoid) for confirmation, and 5% (v/v) sheep blood agar plates (Oxoid) to test for a haemolytic reaction. Further characterization included Gram staining, catalase test, determination of motility at 25°C, utilization of rhamnose and xylose, and species-level identification using VITEK2 gram-positive (GP) identification cards (bioMérieux, France). Confirmed L. monocytogenes isolates were stored in 20% glycerol at -80°C for further assays.
Quality control strains used during the study were L. monocytogenes (ATCC13932, ATCC19111) and Listeria ivanovii subsp. ivanovii (ATCC19119).
Serotyping of L. monocytogenes isolates was performed with the multiplex PCR method, developed by ANSES (Kerouanton et al., 2013), and a commercially available Listeria Antisera Set according to the manufacturer’s instruction (Denka Seiken, Japan). Briefly, DNA extraction was performed on overnight cultured colonies on 5% sheep blood agar plates (Oxoid) suspended in 100 μL PCR clean water, boiled for 20 minutes and centrifuged at 14 000 g for 1 minute. The supernatant was used in the PCR reaction. Amplification reactions were carried out on a PCRmax thermocycler. PCR products were run in 2% agarose gel and visualized by staining with ethidium bromide. Genoserotyped L. monocytogenes strains from the EURL-LM collection were used as positive controls for PCR serotyping, and ultra purified water as the negative control.
Conventional serotyping for L. monocytogenes was carried out using the commercially available Listeria Antisera (Denka Seiken) according to manufacturer’s instructions for strains belonging to PCR serogroups IIa, IIb, IIc and IVb (Table 1).
Results and Discussion
In the long-distance chain, wild birds, especially migratory birds such as gulls, are an important link in the transmission of various pathogenic microorganisms of public interest (Munster et al., 2007; Literak et al., 2010; Jurinović et al., 2014).
In this study, a total of 390 samples of gulls’ cloacal swabs were analysed from 2018 to 2020 for the presence of Listeria spp. Among them, 97 (24.9%) samples showed growth of the presumptive colonies on both agars already after the first enrichment step, and a total of 108 Listeria strains were isolated from these samples (Table 2).
Of the total 144 cloacal swabs examined in 2018, 42 (29.2%) contained bacteria of the genus Listeria, and 49 bacterial strains were isolated. Of these isolates, 27 (55.1%) belonged to the species L. innocua, 21 (42.9%) to L. monocytogenes, and one (2.0%) was identified as L. seeligeri. The simultaneous presence of two different species was found in seven (17.0%) positive samples, with the combination of L. innocua and L. monocytogenes proven in six samples (14.3%), and of L. monocytogenes and L. seeligeri in one (2.0%) positive swab sample examined in 2018.
In 2019, a total of 106 gulls’ cloacal swabs were taken and examined, of which 27 (25.5 %) contained bacteria of the genus Listeria. The presence of L. innocua was found in 13 (48.1%) positive samples, L. monocytogenes in nine (33.3%), L. ivanovii in two (7.4%) and L. welshimeri in one (3.7%) sample. In the remaining two (7.4%) of the total number of positive swab samples, the simultaneous presence of different species was determined, where one was a combination of L. innocua and L. monocytogenes (3.7%) and the second contained a combination of three species L. innocua, L. monocytogenes and L. welshimeri (3.7%).
A total of 140 cloacal swabs were examined in 2020, of which 28 (20.0%) contained bacteria of the genus Listeria.
Out of 29 isolates of Listeria spp., L. innocua was proven for 13 (46.4%) isolates, 11 strains (39.3%) belonged to L. monocytogenes, and three isolates were identified as L. welshimeri (10.7%). The presence of two different species – L. innocua and L. monocytogenes – was detected in the remaining isolated strain (3.6%).
Among the 108 strains of isolated Listeria spp., the most prevalent species was L. innocua (56 isolates, 51.9%), followed by L. monocytogenes (44 isolates, 40.7%), L. welshimeri (5 isolates, 4.6%), L. ivanovii (2 isolates, 1.9%) and L. seeligeri (1 isolate, 0.9%).
The average occurrence of L. monocytogenes in gulls in this study was 11.3%, with the highest prevalence in 2018 (14.6%), and decreased occurrence in 2019 and 2020 (10.4% and 8.6%, respectively).
These results are concordant with earlier studies in wild birds that reported a prevalence of L. monocytogenes from 0 to 36% (Yoshida v., 2000; Hellström et al., 2007; Gan et al., 2019). Fenlon (1985) found a higher prevalence of L. monocytogenes in gulls feeding around sewage (15.2%) than those feeding elsewhere (4.5%), and our results from a landfill are in the middle of these values. While the prevalence of L. monocytogenes is similar to those from previous studies, L. innocua was dominant in our dataset with more than half of all isolates obtained and a prevalence of 14.4%, which is higher than reported by Fenlon (1985) and Duarte et al. (2002) who found it in 4.5% and 5.3% of gull samples, respectively, but in concordance with Quessy and Messier (1992) who found it in 13.6% of gull samples. It is estimated that more than 10,000 gulls feed at this landfill in Zagreb during the winter, and these gulls have been proven to original from throughout Europe (Jurinović, 2018). Therefore, this may be significant for the transmission of Listeria species.
Multiplex PCR was performed on serogroup 44 L. monocytogenes isolates, and the results showed that 43.2% of isolates belong to the 1/2a-3a serogroup, 43.2% to the 1/2b-3b-7 serogroup and 13.6% to the 4b-4d-4e serogroup.
Conventional serotyping was also performed on these 44 isolates that were subtyped into three serotypes: serotypes 1/2a and 1/2b were predominant and each included 19 isolates (43.2%), while the six remaining strains (13.6%) belonged to serotype 4b.
Table 3 shows the concordance in the classification of isolates into serotypes using PCR primers with the classification based on conventional serotyping for all 44 L. monocytogenes tested strains. With such a high proportion of serotypes potentially causing human disease, gulls feeding at this landfill site should be monitored for the presence of Listeria species.
To the best of our knowledge, this is the first report investigating the presence of L. monocytogenes and other Listeria spp. in wild birds in Croatia.
We would like to thank the “Jaki Dečki” ringing group for catching gulls. This research was funded by the Croatian Veterinary Institute and ZGOS Ltd.
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Pojavnost bakterije Listeria monocytogenes i drugih vrsta roda Listeria u galebova koji se hrane na zagrebačkom odlagalištu otpada, Hrvatska
Dr. sc. Andrea HUMSKI, dr. med. vet., znanstvena savjetnica, naslovna docentica, dr. sc. Mario MITAK, dr. med. vet., znanstveni savjetnik u trajnom zvanju, dr. sc. Marina MIKULIĆ, dr. med. vet., znanstvena suradnica, dr. sc. Dora STOJEVIĆ, dr. med. vet., poslijedoktorandica, Hrvatski veterinarski institut, Zagreb, Hrvatska; dr. sc. Borka ŠIMPRAGA, dr. med. vet., viša znanstvena suradnica, mr. spec. Fani KRSTULOVIĆ, dr. med. vet., stručna savjetnica, Biljana JEČMENICA, mag. oecol. et prot. nat., dr. sc. Tajana AMŠEL ZELENIKA, dr. med. vet., znanstvena suradnica, dr. sc. Luka JURINOVIĆ, mag. biol., Hrvatski veterinarski institut – podružnica Centar za peradarstvo, Zagreb, Hrvatska
Za galebove je kao divlje ptice selice poznato da mogu na velike udaljenosti širiti različite patogene. Cilj je ovog istraživanja bio odrediti prevalenciju različitih vrsta roda Listeria u populaciji galebova koji se hrane na Zagrebačkom odlagalištu otpada. Sveukupno je pretraženo 390 galebova triju vrsta: galeb klaukavac – Larus michahellis; riječni galeb – L. ridibundus i burni galeb – L. canus. Najzastupljenija vrsta bila je L. innocua (14,4 %), dok je L. monocytogenes dokazana u 11,3 % ispitanih uzoraka. Prisutnost ostalih vrsta zastupljena je u manjem postotku i to L. welshimeri (1,3 %), L. ivanovii (0,5 %) i L. seeligeri (0,3 %). Serotipizacija izolata L. monocytogenes provedena je molekularnim i konvencionalnim metodama, a većina izolata pripadala je serotipovima 1/2a i 1/2b. Prema našem saznanju, ova studija donosi prve rezultate istraživanja prisutnosti L. monocytogenes i drugih vrsta roda Listeria u divljih ptica u Hrvatskoj.
Ključne riječi: divlje ptice, zoonoza, Listeria monocytogenes