Accepted Manuscript Title: Microbiological survey of birds of prey pellets Author: Ludovico Dipineto Luigi Maria De Luca Bossa Antonino Pace Tamara Pasqualina Russo Antonio Gargiulo Francesca Ciccarelli Pasquale Raia Vincenzo Caputo Alessandro Fioretti PII: DOI: Reference:
S0147-9571(15)00030-2 http://dx.doi.org/doi:10.1016/j.cimid.2015.05.001 CIMID 1009
To appear in: Received date: Revised date: Accepted date:
30-10-2014 2-5-2015 9-5-2015
Please cite this article as: Dipineto L, Bossa LMDL, Pace A, Russo TP, Gargiulo A, Ciccarelli F, Raia P, Caputo V, Fioretti A, Microbiological survey of birds of prey pellets, Comparative Immunology, Microbiology and Infectious Diseases (2015), http://dx.doi.org/10.1016/j.cimid.2015.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Microbiological survey of birds of prey pellets
Highlights
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A microbiological survey of pellets produced by birds of prey was performed
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Pellets of birds of prey contained a wide range of bacteria, also zoonotic
Birds of prey may serve as asymptomatic carriers of pathogenic bacteria
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Birds of prey may spread pathogenic bacteria through their pellets
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Microbiological survey of birds of prey pellets Running Title: Microbiological survey of pellets
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Ludovico Dipineto1*, Luigi Maria De Luca Bossa1,2, Antonino Pace1, Tamara Pasqualina Russo1,
*
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Antonio Gargiulo1, Francesca Ciccarelli3, Pasquale Raia3, Vincenzo Caputo2, Alessandro Fioretti1
Corresponding author. Mailing address: Dipartimento di Medicina Veterinaria e Produzioni
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Animali, Università di Napoli Federico II, via della Veterinaria 1, 80137, Napoli, Italy. Phone: +39
Department of Veterinary Medicine and Animal Productions, Università di Napoli Federico II, via
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1
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0812536277. Fax: +39 0812536280. E-mail: [email protected]
F. Delpino 1, 80137, Napoli, Italy.
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3
Regional Reference Center of Urban Veterinary Hygiene (CRIUV), Napoli, Italy. Wildlife Rescue and Rehabilitation Center “CRAS-Frullone”, Via M. Rocco di Torrepadula,
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Napoli, Italy.
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Abstract A microbiological survey of 73 pellets collected from different birds of prey species housed at the Wildlife Rescue and Rehabilitation Center of Napoli (southern Italy) was performed. Pellets were
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analyzed by culture and biochemical methods as well as by serotyping and polymerase chain reaction. We isolated a wide range of bacteria some of them also pathogens for humans (i.e.
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Salmonella enterica serotype Typhimurium, Campylobacter coli, Escherichia coli O serogroups). This study highlights the potential role of birds of prey as asymptomatic carriers of pathogenic
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bacteria which could be disseminated in the environment not only through the birds of prey faeces
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but also through their pellets.
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Key words: Birds of prey; pellets; zoonotic bacteria, Salmonella spp., Campylobacter spp.
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1. Introduction
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Pellets are regurgitated oblong masses of the undigested remains of prey ingested by a bird of prey. Pellets usually consist of fur, bones, claws, and teeth. Pellet formation occurs within the gizzard.
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Muscular contractions then push the pellet up into the lower esophagus. From there, antiperistaltic waves move the pellet toward the oropharynx where it is expelled. The volume, appearance and timing of pellets varies according to diet fed and, to a lesser extent, the individual bird [1]. Because pellets are characterized by survival of a high proportion of skeletal material they were used to collect information in taphonomic, environmental and biological studies [2-4]. Furthermore, it is possible that viable pathogens such as viruses and bacteria may be present in pellets becoming themselves a risk to human health. In this respect, outbreaks of salmonellosis associated with dissection of owl pellets were reported at two elementary schools by Smith et al. [5] in the USA. In light of the above, the present study was undertaken with the aim to perform a microbiological survey of birds of prey pellets with specific reference to zoonotic bacteria. 3
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2. Materials and Methods 2.1. Sampling During the period January 2012–January 2014, a total of 73 birds of prey housed at the Wildlife
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Rescue and Rehabilitation Center (WRRC) of Napoli (southern Italy) was examined. Birds belonged to several avian species. In particular, there were n=26 Common kestrel (Falco
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tinnunculus), n=14 Peregrine falcon (Falco peregrinus), n=13 Common buzzard (Buteo buteo), n=6 Eurasian sparrowhawk (Accipiter nisus), n=5 Barn owl (Tyto alba), n=4 Tawny owl (Strix aluco),
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n=2 Eurasian eagle-owl (Bubo bubo), n=2 Short-eared owl (Asio flammeus), n=1 Short-toed eagle (Circaetus gallicus). Each bird of prey was temporarily placed in a cardboard box. For each bird,
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pellet was collected at the time of regurgitation by using a sterile surgical drape placed on the base
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of the cardboard box. For the majority of birds, sample collection was on the day of admission (before treatment administration) and before housing in the hospitalization cage or aviary. Each
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pellet was weighed and five equal size samples were collected from the innermost part by using
Use guidelines.
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2.2. Isolation procedures
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sterile lancet. Bird-handling procedures were performed according to the Office of Animal Care and
The aliquots of pellets were inoculated in buffered peptone water (BPW), Campylobacter-selective enrichment broth (CSEB), cooked meat medium (CMM), modified tryptone soya broth (MTSB), phosphate buffered saline (PBS). Samples inoculated into BPW were incubated at 37 °C for 24 h and then were placed into Rappaport-Vassiliadis broth (RV) as well as plated onto Columbia blood agar base (CBA; Oxoid), Pseudomonas cetrimide agar (PCA; Oxoid), MacConkey agar (MCA; Oxoid) and Baird-Parker agar (BPA; Oxoid). Samples inoculated into MTSB were incubated at 37 °C for 24 h and then plated onto sorbitol MacConkey agar (SMCA; Oxoid) supplemented with cefixime-tellurite (Oxoid) and chromogenic E. coli O157 Agar (CEOA; Biolife Italiana S.r.l., Milan, Italy). Samples inoculated into CSEB were incubated in microaerobic atmosphere (oxygen 4
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level of 8-9% and carbon dioxide level below 8%) provided by CampyGen (Oxoid) at 42 °C for 48 h and then plated onto Campylobacter blood-free selective agar (CBFA; Oxoid). Samples inoculated into CMM were incubated in anaerobic atmosphere (oxygen level below 0.5% and
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carbon dioxide level between 9% and 13%) provided by AnaeroGen (Oxoid) at 37 °C for 24 h and then streaked onto anaerobe basal agar (ABA; Oxoid). Samples inoculated into PBS were incubated
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at 4 °C for 14 days and then streaked onto Yersinia selective agar base (cefsulodin-irgasan-
novobiocin, CIN Agar; Oxoid) with incubation at 30 °C for 24-48 h. The CBA, PCA, MCA,
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SMCA, CEOA and BPA plates were incubated at 37 °C for 24–48 h, whereas the RV broths were incubated at 42 °C for 24-48 h and then plated onto both xylose lysine desoxycholate agar (XLD)
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and brilliant green agar (BGA), the CBFA plates were incubated microaerobically at 42 °C,
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whereas ABA plates were anaerobically incubated at 37 °C for 48 h and checked daily for a further week before discarding.
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2.3. Identification procedures
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All strains isolated were primarily identified, selecting 2-3 colonies from plates, on the basis of their colonial morphology, Gram and acid-fastness characteristics, growth requirements, motility
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tests, pigment production, tube coagulase test, and standard conventional biochemical and phenotypic tests. The isolates were confirmed by using API 20 E, API 20 NE systems (bioMerieux, Mercy-l’Etoile, France) and RapID ANA II, RapID NF PLUS, RapID STAPH PLUS Identification Systems (Oxoid). E. coli isolates were serogrouped with antisera poly- and monospecific (Sifin), whereas Salmonella isolates were serotyped according to the Kauffman-White scheme in collaboration with the OIE National Reference Laboratory for Salmonella (IZSVe, Legnaro, Italy). Campylobacter isolates were identified by PCR as reported by Gargiulo et al. [6]. 2.4. Antimicrobial susceptibility testing All isolates were submitted to antimicrobial susceptibility testing using the disc diffusion method. As there are not yet CLSI interpretative criteria for susceptibility breakpoints (disk diffusion) for C. 5
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coli and anaerobic bacteria, antimicrobial susceptibility testing was not performed for these microorganisms. The antimicrobials tested were amoxycillin/clavulanic acid (AMC; 30 μg), tetracycline (TE; 30 μg), ceftazidime (CAZ; 30 μg), gentamicin (CN; 10 μg), enrofloxacin (ENR; 5
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μg), sulfamethoxazole-trimethoprim (SXT; 23.75/1.25 μg) and ciprofloxacin (CIP; 5 μg). In order to evaluate the presence of methicillin-resistant Staphylococcus species, oxacillin (1 μg) and
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cefoxitin (30 μg) disks were also used for Staphylococcus spp. isolates. The inhibition zones were measured and scored as susceptible, intermediate and resistant according to the Clinical and
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Laboratory Standards Institute (CLSI) documents [7-11]. The breakpoints for A. xyloxidans and A. faecalis were those reported for Burkholderia spp. and P. aeruginosa [7-11]. When an antimicrobial
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molecule for a specific agent was not present in the CLSI documents, a similar antimicrobial
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molecule of the same class was used. Specifically, amoxycillin/clavulanic acid breakpoints used for A. xyloxidans and A. faecalis were those reported by CLSI for ticarcillin/clavulanic acid of P.
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aeruginosa [10]. Ceftazidime breakpoints for S. aureus and coagulase-negative Staphylococcus spp.
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were those reported by CLSI for ceftaroline [10] and cefoxitin [8] breakpoints, respectively. Finally, enrofloxacin breakpoints for Enterobacteriaceae were those suggested in CLSI standards
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for chicken and turkeys [8]. In contrast, enrofloxacin breakpoints for the remaining bacteria are not available, thus the corresponding ciprofloxacin breakpoints reported by CLSI [8,10] were used. Breakpoints used for disk diffusion were summarized in table 2. S. aureus ATCC 25923 and E. coli ATCC 25922 were used as control strains.
3. Results
Pellets of birds of prey tested in the present study contained a wide range of bacteria both Gramnegative and Gram-positive species. Different bacterial species were simultaneously recovered from each pellet. Among Gram-negative isolates, E. coli was detected from 48/73 (65.8%; 95% Confidence Interval [CI] = 53.6 – 76.2%) pellets examined and serogrouped as O26 (n=8), O55 6
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(n=2), O103 (n=14), O145 (n=6), O164 (n=4); the remaining strains were identified as generic E. coli. Salmonella spp. was isolated from 2/73 (2.7%; 95% CI = 0.5 – 10.4%) pellets examined and serotyped as Salmonella enterica serovar Typhimurium. Thermotolerant Campylobacter spp. was
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isolated from 26/73 (35.6%; 95% CI = 25.0 – 47.8%) pellets examined and identified as C. coli. Furthermore, Enterobacter cloacae was isolated from 18/73 (24.7%; 95% CI = 15.6 – 36.4%) and
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E. amnigenus from 20/73 (27.4%; 95% CI = 17.9 – 39.3%) samples, Citrobacter freundii (API code 1604572) was isolated from 24/73 (32.9%; 95% CI = 22.6 – 45.0%), C. brakii (API code 0704553)
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from 2/73 (2.7%; 95% CI = 0.5 – 10.4%), and C. youngae (API code 1604512) from 2/73 (2.7%; 95% CI = 0.5 – 10.4%) samples, Klebsiella pneumoniae was isolated from 12/73 (16.4%; 95% CI =
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9.1 – 27.4%) pellets, Achromobacter xylosoxidans was isolated from 10/73 (13.7%; 95% CI = 7.1 –
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24.2%) and Alcaligenes faecalis was isolated from 2/73 (2.7%; 95% CI = 0.5 – 10.4%) pellets examined. In contrast, Pseudomonas spp. and Yersinia spp. were never recovered. With respect to
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Gram-positive isolates, Staphylococcus spp. was detected from 64/73 (87.7%; 95% CI = 77.4 –
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93.9%) pellet samples and identified as S. aureus (n=26); the remaining strains were identified as coagulase-negative staphylococci. Methicillin-resistant Staphylococcus species were not detected.
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Low recovery rates for anaerobic bacteria were observed. Specifically, Fusobacterium necrophorum was isolated from 7/73 (9.6%; 95% CI = 4.3 – 19.3%) samples, Clostridium perfringens was isolated from 4/73 (5.5%; 95% CI = 1.8 – 14.2%) and Bacteroides species was isolated from 3/73 (4.1%; 95% CI = 1.1 – 12.3%) pellet samples analysed. Regarding the antimicrobial susceptibility, Gram-negative isolates showed high percentage of susceptibility to enrofloxacin (ENR) and ciprofloxacin (CIP), followed by sulfamethoxazole/trimethoprim (SXT); Gram-positive isolates showed high susceptibility to enrofloxacin (ENR) and ciprofloxacin (CIP), followed by gentamicin (CN). Results of microbiological findings and antimicrobial susceptibility test are summarized in Table 1. 4. Discussion 7
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There has been a recent increase in studies using pellets for different purposes such as taphonomic, environmental and biological studies [2-4]. Nevertheless, microbiological studies on pellets are scant and referred to anecdotal reports [5,12]. When interpreting microbiologic cultures in clinically
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affected animals, it is important to differentiate pathogenic and commensal bacteria. All animals carry commensal bacteria that are usually nonpathogenic and can protect the body from
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colonization by pathogenic organisms. Regarding the bacterial flora of birds of prey pellets isolated in the present study, the most frequently isolated organisms were bacteria belonging to the
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Enterobacteriaceae family followed by Staphylococcus species and anaerobic bacteria. In our study, twelve genera of bacteria were identified and the majority are considered to be pathogens (i.e. S.
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Typhimurium, C. coli, E. coli O serogroups) or opportunistic pathogens (e.g. K. pneumonia, S.
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aureus, C. perfringens) for humans. The isolation of S. Typhimurium is an interesting finding and, although infrequently isolated with a low percentage (2.7%), this may confirm pellets as a potential
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source of infection for humans. In fact, in a study conducted by Smith et al. [5], S. Typhimurium
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was responsible of outbreaks occurred at two elementary schools associated with dissection of owl pellets. E. coli was isolated with a prevalence of 65.8% and the majority of isolates were
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serogrouped as potential shigatoxin-producing strains (i.e. O26, O103, O145). With respect to thermotolerant Campylobacter isolation, C. coli was the only species identified with a prevalence of 35.6%. In literature, similar results were observed in game birds [13,14] in which C. coli was recovered with a prevalence higher than C. jejuni. Nevertheless, more species of nonthermotolerant Campylobacter may have been found by incubating at 37° C. Regarding the other bacteria isolated in the present study, the majority was reported as emerging nosocomial pathogens in several studies. In particular, Staphylococcus spp., Citrobacter spp., K. pneumoniae and E. cloacae were recorded as responsible of both urinary tract infections in hospitalized patients [15] and cases of neonatal sepsis [16], whereas A. xylosoxidans and A. faecalis
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are considered emerging infectious bacterial species that can affect immunosuppressed patients as well as patients with cancer [17]. It is not possible to speculate regarding the source of bacteria recovered in the pellets examined in
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the present study. However, feeding may be the most plausible source of infection. In fact, in a study conducted by Kirkwood et al. [18], the same S. Enteritidis phage type was isolated both from
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birds of prey faecal samples and from a pool of chicks used for their diet. Similar results were
obtained from a study conducted by Smith et al. [5] who isolated the same S. Typhimurium phage
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type both from owl samples and from chicks used to feed them. It is therefore conceivable that diet of birds of prey may also have affected the bacterial flora of pellets collected from birds of prey
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examined in the present study. Various studies were conducted to evaluate the presence of
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pathogenic bacteria (i.e. Campylobacter spp., Salmonella spp.) in the faeces of these avian species [19-21] suggesting their role as carrier of zoonotic agents. The present study, therefore, highlights
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the potential role of birds of prey as asymptomatic carriers of pathogenic bacteria which could be
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disseminated in the environment not only through the birds of prey faeces but also through their pellets. Although no statistical analyses were performed, the bird of prey species with a greater
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diversity of bacteria isolated were represented by Common kestrel, Peregrine falcon and Common buzzard. It would be useful to establish whether the birds of prey should be regarded as ‘‘target species” of bacterial infections or as animals that, conversely, deteriorate the sanitary and epidemiologic conditions of the wild environment by spreading of zoonotic agents. In conclusion, until more is known about the epidemiology and prevention of these infections in birds of prey, caution should be exercised in translocation, husbandry, and human contact with these animals. In fact, although wildlife disease outbreaks have often been underreported in the broader context of global epidemiology, birds of prey may serve as a reservoir of pathogens for domestic animal and human health acting at the animal-human-ecosystem interface.
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Conflict of interest The authors have no conflict of interest to declare
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Acknowledgements The authors would like to thank the OIE National Reference Laboratory for Salmonella (IZSVe,
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Legnaro, Italy) for serotyping of Salmonella strains.
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Passerine Birds, Gloucester UK: British Small Animal Veterinary Association; 2008, p. 190-201.
2. Terry RC. Owl Pellet Taphonomy: A Preliminary Study of the Post-Regurgitation Taphonomic History
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of Pellets in a Temperate Forest. Palaios 2004; 19:497-506.
3. Scheibler DR, Christoff AU. Habitat associations of small mammals in southern Brazil and use of
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images. Ornis Fennica 2007; 84:21-31.
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4. Tornberg R, Reif V. Assessing the diet of birds of prey: a comparison of prey items found in nests and
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5. Smith KE, Anderson F, Medus C, Leano F, Adams J. Outbreaks of salmonellosis at elementary schools
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8. CLSI. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals; Second Informational Supplement. CLSI document VET01-S2. Wayne PA: Clinical and Laboratory Standards Institute, 2013. 11
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10. CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational
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Supplement. CLSI document M100-S25. Wayne PA: Clinical and Laboratory Standards Institute, 2015.
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11. CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Tenth Edition. CLSI document M07-A10. Wayne PA: Clinical and Laboratory
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Standards Institute, 2015.
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12. Dobrokhotov BP, Meshcheryakova IS. Detection of enzootic territories and exploration of tularemia epizootics in different types of natural foci of this infection by serological examination of bird pellets
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and the excrements of beasts of prey. J Hyg Epidemiol Microbiol Immunol 1980; 24:97-103.
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13. Dipineto L, Gargiulo A, De Luca Bossa LM, Rinaldi L, Borrelli L, Santaniello A, et al. Prevalence of
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thermotolerant Campylobacter in partridges (Perdix perdix). Lett Appl Microbiol 2009; 49:351-353.
14. Dipineto L, Gargiulo A, De Luca Bossa LM, Rinaldi L, Borrelli L, Menna LF, et al. Prevalence of thermotolerant Campylobacter in pheasants (Phasianus colchicus). Avian Pathol 2008; 37:507-508.
15. Qian L, Camara T, Taylor JK, Jones KW. Microbial uropathogens and their antibiotic resistance profile from hospitalized patients in Central Alabama. Clin Lab Sci 2012; 25:206-11.
16. Sharma P, Kaur P, Aggarwal A. Staphylococcus aureus- the predominant pathogen in the neonatal ICU of a tertiary care hospital in Amritsar, India. J Clin Diagn Res 2013; 7:66-69.
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17. Aisenberg G, Rolston KV, Safdar, A. Bacteremia caused by Achromobacter and Alcaligenes species in 46 patients with cancer (1989-2003). Cancer 2004; 101: 2134-2140.
by carnivorous animals fed on day-old chicks. Vet Rec 1994; 134(26):683.
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18. Kirkwood JK, Cunningham AA, Macgregor SK, Thornton SM, Duff JP. Salmonella enteritidis excretion
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19. Palmgren H, Broman T, Waldenström J, Lindberg P, Aspán A, Olsen B. Salmonella Amager,
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Campylobacter jejuni, and urease-positive thermophilic Campylobacter found in free-flying peregrine
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falcons (Falco peregrinus) in Sweden. J Wildl Dis 2004; 40(3):583-587.
20. Molina-Lopez RA, Valverdú N, Martin M, Mateu E, Obon E, Cerdà-Cuéllar M, Darwich L. Wild raptors
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as carriers of antimicrobial-resistant Salmonella and Campylobacter strains. Vet Rec 2011; 168(21):565.
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Fioretti A. Campylobacter spp. and birds of prey. Avian Dis 2014; 58(2):303-305.
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Table 1. Bacteria isolated from pellets collected from 73 birds of prey and related results of antimicrobial susceptibility test
Antimicrobial susceptibility test (%)a AMC
TE
CAZ
CN
ENR
SXT
CIP
30 µgb
30 µgb
30 µgb
10 µgb
5 µgb
25 µgb
5 µgb
20
85
80
85
75
70
80
70
Citrobacter freundii
24
50
75
50
83
83
67
83
Enterobacter cloacae
18
100
83
83
72
72
89
72
Citrobacter youngae
2
0
100
0
100
100
100
100
Citrobacter brakii
2
0
100
50
100
100
100
100
Salmonella Typhimurium
2
50
0
50
0
100
50
100
Klebsiella pneumoniae
12
17
83
42
75
83
67
83
Escherichia coli
48
92
73
92
94
94
63
92
Achromobacter xyloxidans
10
50
50
80
20
30
80
30
50
50
50
0
100
50
100
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Alcaligenes faecalis
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Enterobacter amnigenus
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Gram-negative bacteria
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Number tested
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Bacteria
Gram-positive bacteria coagulase-negative Staphylococcus spp.
38
58
79
53
84
82
76
79
Staphylococcus aureus
26
38
58
42
73
77
65
81
a
Percentage of susceptibility Concentration of the disc used for testing AMC = amoxycillin/clavulanic acid, TE = tetracycline, CAZ = ceftazidime, CN = gentamicin, ENR = enrofloxacin, SXT = sulfamethoxazole/trimethoprim, CIP = ciprofloxacin b
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1
Table 2. Antimicrobial breakpoints used for the interpretation of susceptibility and resistance of bacteria isolated from birds of prey pellets. Antimicrobial tested and related breakpoints (zone diameter in mm) AMC
TE
CAZ
S
I
R
S
I
R
S
≥18
14-17
≤13
≥15
12-14
≤11
≥21
≥24b
16-23b
≤15b
≥19
15-18
≤14
coagulase-negative Staphylococcus spp.
≥20
-
≤19
≥19
15-18
Staphylococcus aureus
≥20
-
≤19
≥19
15-18
Gram-negative bacteria
I
CN
R
Citrobacter freundii Enterobacter cloacae Citrobacter youngae
Alcaligenes faecalis
a
2 3 4 5 6 7 8 9
I
R
S
I
R
S
I
R
S
I
R
13-14
≤12
≥23
17-22
≤16
≥16
11-15
≤10
≥31
21-30
≤20
≥21
18-20
≤17
≥15
13-14
≤12
≥21c
16-20c
≤15c
≥16
11-15
≤10
≥21
16-20
≤15
≤14
≥25d
-
≤24d
≥15
13-14
≤12
≥21e
16-20e
≤15e
≥16
11-15
≤10
≥21
16-20
≤15
≤14
≥24f
21-23f
≤20f
≥15
13-14
≤12
≥21e
16-20e
≤15e
≥16
11-15
≤10
≥21
16-20
≤15
ed
Ac
Gram-positive bacteria
CIP
≥15
ce pt
Achromobacter xyloxidansa
SXT
≤17
Escherichia coli
Salmonella Typhimurium
ENR
18-20
Citrobacter brakii
Klebsiella pneumoniae
S
M an
Enterobacter amnigenus
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Bacteria
AMC = amoxycillin/clavulanic acid, TE = tetracycline, CAZ = ceftazidime, CN = gentamicin, ENR = enrofloxacin, SXT = sulfamethoxazole/trimethoprim, CIP = ciprofloxacin, S = susceptible, I = intermediate, R = resistant a = the breakpoints for A. xyloxidans and A. faecalis were those of Burkholderia spp. and P. aeruginosa b = the breakpoints used for amoxycillin/clavulanic acid were those reported for ticarcillin/clavulanic acid of P. aeruginosa c = the breakpoints used for enrofloxacin were those reported for ciprofloxacin of P. aeruginosa d = the breakpoints used for ceftazidime were those reported for cefoxitin of coagulase-negative Staphylococcus spp. e = the breakpoints used for enrofloxacin were those reported for ciprofloxacin of Staphylococcus spp. f = the breakpoints used for ceftazidime were those reported for ceftaroline of Staphylococcus aureus
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S0147-9571(15)00030-2 http://dx.doi.org/doi:10.1016/j.cimid.2015.05.001 CIMID 1009
To appear in: Received date: Revised date: Accepted date:
30-10-2014 2-5-2015 9-5-2015
Please cite this article as: Dipineto L, Bossa LMDL, Pace A, Russo TP, Gargiulo A, Ciccarelli F, Raia P, Caputo V, Fioretti A, Microbiological survey of birds of prey pellets, Comparative Immunology, Microbiology and Infectious Diseases (2015), http://dx.doi.org/10.1016/j.cimid.2015.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Microbiological survey of birds of prey pellets
Highlights
ip t
A microbiological survey of pellets produced by birds of prey was performed
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Pellets of birds of prey contained a wide range of bacteria, also zoonotic
Birds of prey may serve as asymptomatic carriers of pathogenic bacteria
Ac ce p
te
d
M
an
us
Birds of prey may spread pathogenic bacteria through their pellets
1
Page 1 of 15
Microbiological survey of birds of prey pellets Running Title: Microbiological survey of pellets
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Ludovico Dipineto1*, Luigi Maria De Luca Bossa1,2, Antonino Pace1, Tamara Pasqualina Russo1,
*
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Antonio Gargiulo1, Francesca Ciccarelli3, Pasquale Raia3, Vincenzo Caputo2, Alessandro Fioretti1
Corresponding author. Mailing address: Dipartimento di Medicina Veterinaria e Produzioni
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Animali, Università di Napoli Federico II, via della Veterinaria 1, 80137, Napoli, Italy. Phone: +39
Department of Veterinary Medicine and Animal Productions, Università di Napoli Federico II, via
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1
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0812536277. Fax: +39 0812536280. E-mail: [email protected]
F. Delpino 1, 80137, Napoli, Italy.
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3
Regional Reference Center of Urban Veterinary Hygiene (CRIUV), Napoli, Italy. Wildlife Rescue and Rehabilitation Center “CRAS-Frullone”, Via M. Rocco di Torrepadula,
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Napoli, Italy.
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Abstract A microbiological survey of 73 pellets collected from different birds of prey species housed at the Wildlife Rescue and Rehabilitation Center of Napoli (southern Italy) was performed. Pellets were
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analyzed by culture and biochemical methods as well as by serotyping and polymerase chain reaction. We isolated a wide range of bacteria some of them also pathogens for humans (i.e.
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Salmonella enterica serotype Typhimurium, Campylobacter coli, Escherichia coli O serogroups). This study highlights the potential role of birds of prey as asymptomatic carriers of pathogenic
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bacteria which could be disseminated in the environment not only through the birds of prey faeces
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but also through their pellets.
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Key words: Birds of prey; pellets; zoonotic bacteria, Salmonella spp., Campylobacter spp.
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1. Introduction
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Pellets are regurgitated oblong masses of the undigested remains of prey ingested by a bird of prey. Pellets usually consist of fur, bones, claws, and teeth. Pellet formation occurs within the gizzard.
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Muscular contractions then push the pellet up into the lower esophagus. From there, antiperistaltic waves move the pellet toward the oropharynx where it is expelled. The volume, appearance and timing of pellets varies according to diet fed and, to a lesser extent, the individual bird [1]. Because pellets are characterized by survival of a high proportion of skeletal material they were used to collect information in taphonomic, environmental and biological studies [2-4]. Furthermore, it is possible that viable pathogens such as viruses and bacteria may be present in pellets becoming themselves a risk to human health. In this respect, outbreaks of salmonellosis associated with dissection of owl pellets were reported at two elementary schools by Smith et al. [5] in the USA. In light of the above, the present study was undertaken with the aim to perform a microbiological survey of birds of prey pellets with specific reference to zoonotic bacteria. 3
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2. Materials and Methods 2.1. Sampling During the period January 2012–January 2014, a total of 73 birds of prey housed at the Wildlife
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Rescue and Rehabilitation Center (WRRC) of Napoli (southern Italy) was examined. Birds belonged to several avian species. In particular, there were n=26 Common kestrel (Falco
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tinnunculus), n=14 Peregrine falcon (Falco peregrinus), n=13 Common buzzard (Buteo buteo), n=6 Eurasian sparrowhawk (Accipiter nisus), n=5 Barn owl (Tyto alba), n=4 Tawny owl (Strix aluco),
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n=2 Eurasian eagle-owl (Bubo bubo), n=2 Short-eared owl (Asio flammeus), n=1 Short-toed eagle (Circaetus gallicus). Each bird of prey was temporarily placed in a cardboard box. For each bird,
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pellet was collected at the time of regurgitation by using a sterile surgical drape placed on the base
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of the cardboard box. For the majority of birds, sample collection was on the day of admission (before treatment administration) and before housing in the hospitalization cage or aviary. Each
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pellet was weighed and five equal size samples were collected from the innermost part by using
Use guidelines.
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2.2. Isolation procedures
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sterile lancet. Bird-handling procedures were performed according to the Office of Animal Care and
The aliquots of pellets were inoculated in buffered peptone water (BPW), Campylobacter-selective enrichment broth (CSEB), cooked meat medium (CMM), modified tryptone soya broth (MTSB), phosphate buffered saline (PBS). Samples inoculated into BPW were incubated at 37 °C for 24 h and then were placed into Rappaport-Vassiliadis broth (RV) as well as plated onto Columbia blood agar base (CBA; Oxoid), Pseudomonas cetrimide agar (PCA; Oxoid), MacConkey agar (MCA; Oxoid) and Baird-Parker agar (BPA; Oxoid). Samples inoculated into MTSB were incubated at 37 °C for 24 h and then plated onto sorbitol MacConkey agar (SMCA; Oxoid) supplemented with cefixime-tellurite (Oxoid) and chromogenic E. coli O157 Agar (CEOA; Biolife Italiana S.r.l., Milan, Italy). Samples inoculated into CSEB were incubated in microaerobic atmosphere (oxygen 4
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level of 8-9% and carbon dioxide level below 8%) provided by CampyGen (Oxoid) at 42 °C for 48 h and then plated onto Campylobacter blood-free selective agar (CBFA; Oxoid). Samples inoculated into CMM were incubated in anaerobic atmosphere (oxygen level below 0.5% and
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carbon dioxide level between 9% and 13%) provided by AnaeroGen (Oxoid) at 37 °C for 24 h and then streaked onto anaerobe basal agar (ABA; Oxoid). Samples inoculated into PBS were incubated
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at 4 °C for 14 days and then streaked onto Yersinia selective agar base (cefsulodin-irgasan-
novobiocin, CIN Agar; Oxoid) with incubation at 30 °C for 24-48 h. The CBA, PCA, MCA,
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SMCA, CEOA and BPA plates were incubated at 37 °C for 24–48 h, whereas the RV broths were incubated at 42 °C for 24-48 h and then plated onto both xylose lysine desoxycholate agar (XLD)
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and brilliant green agar (BGA), the CBFA plates were incubated microaerobically at 42 °C,
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whereas ABA plates were anaerobically incubated at 37 °C for 48 h and checked daily for a further week before discarding.
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2.3. Identification procedures
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All strains isolated were primarily identified, selecting 2-3 colonies from plates, on the basis of their colonial morphology, Gram and acid-fastness characteristics, growth requirements, motility
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tests, pigment production, tube coagulase test, and standard conventional biochemical and phenotypic tests. The isolates were confirmed by using API 20 E, API 20 NE systems (bioMerieux, Mercy-l’Etoile, France) and RapID ANA II, RapID NF PLUS, RapID STAPH PLUS Identification Systems (Oxoid). E. coli isolates were serogrouped with antisera poly- and monospecific (Sifin), whereas Salmonella isolates were serotyped according to the Kauffman-White scheme in collaboration with the OIE National Reference Laboratory for Salmonella (IZSVe, Legnaro, Italy). Campylobacter isolates were identified by PCR as reported by Gargiulo et al. [6]. 2.4. Antimicrobial susceptibility testing All isolates were submitted to antimicrobial susceptibility testing using the disc diffusion method. As there are not yet CLSI interpretative criteria for susceptibility breakpoints (disk diffusion) for C. 5
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coli and anaerobic bacteria, antimicrobial susceptibility testing was not performed for these microorganisms. The antimicrobials tested were amoxycillin/clavulanic acid (AMC; 30 μg), tetracycline (TE; 30 μg), ceftazidime (CAZ; 30 μg), gentamicin (CN; 10 μg), enrofloxacin (ENR; 5
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μg), sulfamethoxazole-trimethoprim (SXT; 23.75/1.25 μg) and ciprofloxacin (CIP; 5 μg). In order to evaluate the presence of methicillin-resistant Staphylococcus species, oxacillin (1 μg) and
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cefoxitin (30 μg) disks were also used for Staphylococcus spp. isolates. The inhibition zones were measured and scored as susceptible, intermediate and resistant according to the Clinical and
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Laboratory Standards Institute (CLSI) documents [7-11]. The breakpoints for A. xyloxidans and A. faecalis were those reported for Burkholderia spp. and P. aeruginosa [7-11]. When an antimicrobial
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molecule for a specific agent was not present in the CLSI documents, a similar antimicrobial
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molecule of the same class was used. Specifically, amoxycillin/clavulanic acid breakpoints used for A. xyloxidans and A. faecalis were those reported by CLSI for ticarcillin/clavulanic acid of P.
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aeruginosa [10]. Ceftazidime breakpoints for S. aureus and coagulase-negative Staphylococcus spp.
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were those reported by CLSI for ceftaroline [10] and cefoxitin [8] breakpoints, respectively. Finally, enrofloxacin breakpoints for Enterobacteriaceae were those suggested in CLSI standards
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for chicken and turkeys [8]. In contrast, enrofloxacin breakpoints for the remaining bacteria are not available, thus the corresponding ciprofloxacin breakpoints reported by CLSI [8,10] were used. Breakpoints used for disk diffusion were summarized in table 2. S. aureus ATCC 25923 and E. coli ATCC 25922 were used as control strains.
3. Results
Pellets of birds of prey tested in the present study contained a wide range of bacteria both Gramnegative and Gram-positive species. Different bacterial species were simultaneously recovered from each pellet. Among Gram-negative isolates, E. coli was detected from 48/73 (65.8%; 95% Confidence Interval [CI] = 53.6 – 76.2%) pellets examined and serogrouped as O26 (n=8), O55 6
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(n=2), O103 (n=14), O145 (n=6), O164 (n=4); the remaining strains were identified as generic E. coli. Salmonella spp. was isolated from 2/73 (2.7%; 95% CI = 0.5 – 10.4%) pellets examined and serotyped as Salmonella enterica serovar Typhimurium. Thermotolerant Campylobacter spp. was
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isolated from 26/73 (35.6%; 95% CI = 25.0 – 47.8%) pellets examined and identified as C. coli. Furthermore, Enterobacter cloacae was isolated from 18/73 (24.7%; 95% CI = 15.6 – 36.4%) and
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E. amnigenus from 20/73 (27.4%; 95% CI = 17.9 – 39.3%) samples, Citrobacter freundii (API code 1604572) was isolated from 24/73 (32.9%; 95% CI = 22.6 – 45.0%), C. brakii (API code 0704553)
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from 2/73 (2.7%; 95% CI = 0.5 – 10.4%), and C. youngae (API code 1604512) from 2/73 (2.7%; 95% CI = 0.5 – 10.4%) samples, Klebsiella pneumoniae was isolated from 12/73 (16.4%; 95% CI =
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9.1 – 27.4%) pellets, Achromobacter xylosoxidans was isolated from 10/73 (13.7%; 95% CI = 7.1 –
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24.2%) and Alcaligenes faecalis was isolated from 2/73 (2.7%; 95% CI = 0.5 – 10.4%) pellets examined. In contrast, Pseudomonas spp. and Yersinia spp. were never recovered. With respect to
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Gram-positive isolates, Staphylococcus spp. was detected from 64/73 (87.7%; 95% CI = 77.4 –
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93.9%) pellet samples and identified as S. aureus (n=26); the remaining strains were identified as coagulase-negative staphylococci. Methicillin-resistant Staphylococcus species were not detected.
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Low recovery rates for anaerobic bacteria were observed. Specifically, Fusobacterium necrophorum was isolated from 7/73 (9.6%; 95% CI = 4.3 – 19.3%) samples, Clostridium perfringens was isolated from 4/73 (5.5%; 95% CI = 1.8 – 14.2%) and Bacteroides species was isolated from 3/73 (4.1%; 95% CI = 1.1 – 12.3%) pellet samples analysed. Regarding the antimicrobial susceptibility, Gram-negative isolates showed high percentage of susceptibility to enrofloxacin (ENR) and ciprofloxacin (CIP), followed by sulfamethoxazole/trimethoprim (SXT); Gram-positive isolates showed high susceptibility to enrofloxacin (ENR) and ciprofloxacin (CIP), followed by gentamicin (CN). Results of microbiological findings and antimicrobial susceptibility test are summarized in Table 1. 4. Discussion 7
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There has been a recent increase in studies using pellets for different purposes such as taphonomic, environmental and biological studies [2-4]. Nevertheless, microbiological studies on pellets are scant and referred to anecdotal reports [5,12]. When interpreting microbiologic cultures in clinically
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affected animals, it is important to differentiate pathogenic and commensal bacteria. All animals carry commensal bacteria that are usually nonpathogenic and can protect the body from
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colonization by pathogenic organisms. Regarding the bacterial flora of birds of prey pellets isolated in the present study, the most frequently isolated organisms were bacteria belonging to the
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Enterobacteriaceae family followed by Staphylococcus species and anaerobic bacteria. In our study, twelve genera of bacteria were identified and the majority are considered to be pathogens (i.e. S.
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Typhimurium, C. coli, E. coli O serogroups) or opportunistic pathogens (e.g. K. pneumonia, S.
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aureus, C. perfringens) for humans. The isolation of S. Typhimurium is an interesting finding and, although infrequently isolated with a low percentage (2.7%), this may confirm pellets as a potential
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source of infection for humans. In fact, in a study conducted by Smith et al. [5], S. Typhimurium
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was responsible of outbreaks occurred at two elementary schools associated with dissection of owl pellets. E. coli was isolated with a prevalence of 65.8% and the majority of isolates were
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serogrouped as potential shigatoxin-producing strains (i.e. O26, O103, O145). With respect to thermotolerant Campylobacter isolation, C. coli was the only species identified with a prevalence of 35.6%. In literature, similar results were observed in game birds [13,14] in which C. coli was recovered with a prevalence higher than C. jejuni. Nevertheless, more species of nonthermotolerant Campylobacter may have been found by incubating at 37° C. Regarding the other bacteria isolated in the present study, the majority was reported as emerging nosocomial pathogens in several studies. In particular, Staphylococcus spp., Citrobacter spp., K. pneumoniae and E. cloacae were recorded as responsible of both urinary tract infections in hospitalized patients [15] and cases of neonatal sepsis [16], whereas A. xylosoxidans and A. faecalis
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are considered emerging infectious bacterial species that can affect immunosuppressed patients as well as patients with cancer [17]. It is not possible to speculate regarding the source of bacteria recovered in the pellets examined in
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the present study. However, feeding may be the most plausible source of infection. In fact, in a study conducted by Kirkwood et al. [18], the same S. Enteritidis phage type was isolated both from
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birds of prey faecal samples and from a pool of chicks used for their diet. Similar results were
obtained from a study conducted by Smith et al. [5] who isolated the same S. Typhimurium phage
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type both from owl samples and from chicks used to feed them. It is therefore conceivable that diet of birds of prey may also have affected the bacterial flora of pellets collected from birds of prey
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examined in the present study. Various studies were conducted to evaluate the presence of
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pathogenic bacteria (i.e. Campylobacter spp., Salmonella spp.) in the faeces of these avian species [19-21] suggesting their role as carrier of zoonotic agents. The present study, therefore, highlights
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the potential role of birds of prey as asymptomatic carriers of pathogenic bacteria which could be
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disseminated in the environment not only through the birds of prey faeces but also through their pellets. Although no statistical analyses were performed, the bird of prey species with a greater
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diversity of bacteria isolated were represented by Common kestrel, Peregrine falcon and Common buzzard. It would be useful to establish whether the birds of prey should be regarded as ‘‘target species” of bacterial infections or as animals that, conversely, deteriorate the sanitary and epidemiologic conditions of the wild environment by spreading of zoonotic agents. In conclusion, until more is known about the epidemiology and prevention of these infections in birds of prey, caution should be exercised in translocation, husbandry, and human contact with these animals. In fact, although wildlife disease outbreaks have often been underreported in the broader context of global epidemiology, birds of prey may serve as a reservoir of pathogens for domestic animal and human health acting at the animal-human-ecosystem interface.
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Conflict of interest The authors have no conflict of interest to declare
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Acknowledgements The authors would like to thank the OIE National Reference Laboratory for Salmonella (IZSVe,
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Legnaro, Italy) for serotyping of Salmonella strains.
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Table 1. Bacteria isolated from pellets collected from 73 birds of prey and related results of antimicrobial susceptibility test
Antimicrobial susceptibility test (%)a AMC
TE
CAZ
CN
ENR
SXT
CIP
30 µgb
30 µgb
30 µgb
10 µgb
5 µgb
25 µgb
5 µgb
20
85
80
85
75
70
80
70
Citrobacter freundii
24
50
75
50
83
83
67
83
Enterobacter cloacae
18
100
83
83
72
72
89
72
Citrobacter youngae
2
0
100
0
100
100
100
100
Citrobacter brakii
2
0
100
50
100
100
100
100
Salmonella Typhimurium
2
50
0
50
0
100
50
100
Klebsiella pneumoniae
12
17
83
42
75
83
67
83
Escherichia coli
48
92
73
92
94
94
63
92
Achromobacter xyloxidans
10
50
50
80
20
30
80
30
50
50
50
0
100
50
100
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Alcaligenes faecalis
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Enterobacter amnigenus
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Gram-negative bacteria
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Number tested
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Bacteria
Gram-positive bacteria coagulase-negative Staphylococcus spp.
38
58
79
53
84
82
76
79
Staphylococcus aureus
26
38
58
42
73
77
65
81
a
Percentage of susceptibility Concentration of the disc used for testing AMC = amoxycillin/clavulanic acid, TE = tetracycline, CAZ = ceftazidime, CN = gentamicin, ENR = enrofloxacin, SXT = sulfamethoxazole/trimethoprim, CIP = ciprofloxacin b
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Table 2. Antimicrobial breakpoints used for the interpretation of susceptibility and resistance of bacteria isolated from birds of prey pellets. Antimicrobial tested and related breakpoints (zone diameter in mm) AMC
TE
CAZ
S
I
R
S
I
R
S
≥18
14-17
≤13
≥15
12-14
≤11
≥21
≥24b
16-23b
≤15b
≥19
15-18
≤14
coagulase-negative Staphylococcus spp.
≥20
-
≤19
≥19
15-18
Staphylococcus aureus
≥20
-
≤19
≥19
15-18
Gram-negative bacteria
I
CN
R
Citrobacter freundii Enterobacter cloacae Citrobacter youngae
Alcaligenes faecalis
a
2 3 4 5 6 7 8 9
I
R
S
I
R
S
I
R
S
I
R
13-14
≤12
≥23
17-22
≤16
≥16
11-15
≤10
≥31
21-30
≤20
≥21
18-20
≤17
≥15
13-14
≤12
≥21c
16-20c
≤15c
≥16
11-15
≤10
≥21
16-20
≤15
≤14
≥25d
-
≤24d
≥15
13-14
≤12
≥21e
16-20e
≤15e
≥16
11-15
≤10
≥21
16-20
≤15
≤14
≥24f
21-23f
≤20f
≥15
13-14
≤12
≥21e
16-20e
≤15e
≥16
11-15
≤10
≥21
16-20
≤15
ed
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Gram-positive bacteria
CIP
≥15
ce pt
Achromobacter xyloxidansa
SXT
≤17
Escherichia coli
Salmonella Typhimurium
ENR
18-20
Citrobacter brakii
Klebsiella pneumoniae
S
M an
Enterobacter amnigenus
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Bacteria
AMC = amoxycillin/clavulanic acid, TE = tetracycline, CAZ = ceftazidime, CN = gentamicin, ENR = enrofloxacin, SXT = sulfamethoxazole/trimethoprim, CIP = ciprofloxacin, S = susceptible, I = intermediate, R = resistant a = the breakpoints for A. xyloxidans and A. faecalis were those of Burkholderia spp. and P. aeruginosa b = the breakpoints used for amoxycillin/clavulanic acid were those reported for ticarcillin/clavulanic acid of P. aeruginosa c = the breakpoints used for enrofloxacin were those reported for ciprofloxacin of P. aeruginosa d = the breakpoints used for ceftazidime were those reported for cefoxitin of coagulase-negative Staphylococcus spp. e = the breakpoints used for enrofloxacin were those reported for ciprofloxacin of Staphylococcus spp. f = the breakpoints used for ceftazidime were those reported for ceftaroline of Staphylococcus aureus
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