Potential Role of Migratory Quail in Spreading of Some Zoonotic Pathogens in Egypt

Corresponding Author: Dalia H. Mansour Department of Poultry and Rabbit Medicine, Faculty of Veterinary Medicine, Suez Canal University, 41522, Ismailia, Egypt Email: Dalia97.mh@gmail.com Abstract: This study aimed to illustrate the potential role of wild migratory quails in the epidemiology of some zoonotic pathogens in Egypt. Ninety four apparently healthy migratory quails were collected from public markets after being trapped during hunting season from the North Sinai. Oral, cloaca and organs (liver, lung and heart) swabs were collected for bacteriological and viral examination. Bacteriological examination revealed high percentage of lactose fermenter bacteria (48.9%) including E. coli spp. (37.2%) and Salmonella spp. (24.4%). Three out of 12 (25%) of Salmonella spp. Isolates were S. enterica subspecies typhimurium. Other bacteria isolation rate was differed such as Staphylococcusaureus spp. (19.1 ) % , Proteus spp. (7.4%), Listeria spp. (4.2%) and Klepsila spp. (3.1%). E. coli spp. and Salmonella spp. isolates were further examined by API20and PCR techniques. Antibiogram testing against 32 E. coli spp. and 12 Salmonella spp. isolates, revealed multi-drug resistance pattern and high sensitivity to enrofloxacin, nitrofuranation and ofloxacine. Parasitological examination revealed that 35 (37.2%) quails were infected with chewing lice (Phithiraptera spp.). Intestinal infection with Heterakis spp. and/or Ascarida spp. Was detected in 27 (28.7%) quails. All quails were negative for avian influenza virus by direct detection by Rt-PCR for oral and cloaca swabs followed by inoculation in embryonated chicken eggs. In conclusion, the migratory quails might play a potential role in dispersion of zoonosis in Egypt.


Introduction
Migratory quail, known as common quail (Coturnix coturnix), is one of the most migratory birds migrates from Europe to Egypt during autumn. During the hunting season, many thousands of these birds are caught in hunter nets on arrival to Mediterranean shore of the North Sinai, Egypt. As other migratory birds, migratory quail act as possible (biological and/or mechanical) vectors playing role in the ecology and circulation of some zoonotic pathogen threatening human health and domestic animals (Abulreesh et al., 2007;Benskin et al., 2009). These zoonotic pathogens cause losses of efficient production and quality of food of animal origin (Acha and Szyfres, 1987;Stohr and Melsin, 1997;Kahn, 2006). Migratory quails pose a risk of transmission of many zoonotic diseases to hunters or consumers who handle or eat these birds by either direct or indirect contact (Smith, 1999). Moreover, wild birds encourage new health problems in wildlife population to emerge, as well as novel reservoir of zoonotic disease could be transmitted and emerge from one continent to another by migratory birds (Cole et al., 2005).
Escherichia coli and Salmonella are considered as the principal causes of morbidity and mortality, associated with heavy economic losses to the poultry industry. Other zoonotic pathogens such as Staphylococcus spp. and Proteus spp. are considered the most important bacterial pathogen isolated from migratory quails (Mohamed et al., 2001;Effat and Moursi, 2005). Moreover, the development of antimicrobial resistant strains of zoonotic bacteria constitutes a public health risk, increasing risk of treatment failures (Middleton and Ambrose, 2005).
Avian influenza AI viruses constitute global health concern. Susceptibility to AI Viruses (AIV) varies deeply among wild bird and poultry species, as well as their possible role as vectors, intermediate hosts or reservoirs. Quails could play a key role in AIV epidemiology (Bertran et al., 2011). It is proposed that quails may generate new re-assortant influenza viruses (Thontiravong et al., 2012).
This study aimed to evaluate the occurrence of some zoonotic pathogens which could be transmitted by wild quails in Egypt as well as studying the antibiotic resistance of some isolated bacterial strains.

Bird Collection
Ninety four apparently healthy live migratory quails were collected from the public markets in two different geographic locations namely Ber-Elabd and Romana ( Fig. 1) across the shore of Mediterranean sea in the North of Sinai, Egypt during hunting season (5th and 20th September and 5th October 2012). All birds were live trapped through nets before being sold out in the markets. Quails were delivered quickly to the laboratory.

Sample Collection
After being delivered to the laboratory, quails were kept under observation for 24 h for clinical examination. Oral and cloaca swabs were collected in two sets tubes, the first contained 10 mL buffered peptone water for bacterial isolation and the second contained 10 mL of Phosphate-Buffered Saline (PBS) solution with antibiotic for viral isolation. Swabs were collected from organs (heart, liver and lung) under aseptic conditions. Intestines were directly opened during postmortem examination and were examined for presence of adult helminthes.

Isolation and Identification of Bacteria
Swabs pre-enrichment was performed in buffered peptone water (LabM) at a temperature of 37°C for 18-24 h followed by plating onto MacConkeyagar (OXOID, Hampshire, UK) and incubated at 37°C for 24 h. The lactose fermenting colonies suspected to E. coli were reinoculated to Eosin Methylene Blue (EMB) agar. Colonies producing metallic sheen were suspected to E. coli strains.
For Salmonella spp. isolation, pre-enrichment was followed by selective enrichment in Rappaport-Vassiliadis (OXOID, Hampshire, UK) at 42°C for 24 h followed by plating on Xylose, Lysine and Desoxycholate (XLD) (LabM) and incubation at 42°C for 24 h. Suspected E. coli, Salmonella, Proteus spp. and Keilbseilla spp. were biochemically identified according to Edwards and Ewing (1972)? Furthermore, suspected E. coli, Salmonella isolates were confirmed using the API 20 test (bioMe´rieux, France). Multiplex PCR was used for identifying S. Enteric serovars typhimurium and enteritidis.

Isolation and Identification of S. Aureus
Pre-enrichment swabs were plated onto selective Mannitol salt agar (LabM) and cultured at 37°C for 24-48 h. Colonies were identified according to Kloos and Bannerman (1999). S. aureus strain, Designation; NCTC7447/ATCC ®6538P was used as control positive.
L. monocytogenes was isolated by pre-enrichment broth incubated at 30°C for 48 h for followed by plating on Listeria Isolation Media, Oxford (ISO) with selective additives (LabM). Grey colonies with black halo were identified as L. monocytogens after incubation at 37°C for 48 h.

DNA Extraction and PCR Amplifications
Bacterial DNA was extracted from cultured broth by centrifugation at 4°C at 3,000×g for 10 min. The pellet was washed twice with PBS and the cells were resuspended in 800 µL of sterile distilled water and boiled for 10 min. The resulting solution was centrifuged at 3,000×g for 10 min and the supernatant was used as the DNA template. Amplification reactions were carried out with 5 µL of boiled bacterial suspensions, 5 µL of 5X Taq Master Mix/high yield (Jena Bioscience, GMBH, Germany) and two pairs of each primer 50 pmol. Distilled water was added to bring the final volume to 25 µL. E. coli spp. isolates; general primers were used in PCR amplification of 670 pb segment of Glutamate Decarboxylase (GAD) gadA/B gene according to McDaniels et al. (1996). Positive microbiological Salmonella were confirmed and serotyped as typhymurium and enteritidis by using multiplex PCR using three sets of primers were used for PCR as described previously by Alvarez et al. (2004). S. aureus isolates were confirmed by amplification of16SrRNA gene according to Monday and Bohatch (1999) that generated a 228-bp amplicon during the amplification process. PCR conditions were started by initial denaturation step of 2 min at 95°C, followed by 30 cycles of 95°C for 30 min, 57-60°C for 30 s and 72°C for 30 sec. The reaction was terminated with 10 min incubation at 72°C. The PCR reaction products were subjected to electrophoresis in a 1.5% agarose gel, stained with ethidium bromide and visualized and photographed under UV light. Products of the appropriate sizes were identified by comparisons with a 100 bp DNA ladder (Gibco). In each PCR run, a non-template control was included to detect possible external DNA contamination and control positive were used for confirmation.

Parasitological Examination
The birds were examined for external parasite and then necropsied for examining of intestine for helminthes parasites. Adult helminthes detected by naked eye were individually recovered, fixed and stored for later identification.

Influenza Virus Isolation and Identification
Oral and cloaca swabs were detected by direct by RT-PCR and by inoculation in 10-day-oldembryonated specific-pathogen-free SPF eggs through allantoic sac route. The inoculated eggs were held at 37°C and candled daily for 5 days. The allantoic fluid was collected and tested by RT-PCR. Swabs were tested for the presence of influenza A viruses by detection of the Matrix (M) gene, using a viral RNA extraction kit (QIAamp, QIAGEN, Germany) and a one-step real-time RT-PCR kit (Quantitect, QIAGEN, Germany). The test was conducted in a Stratagene MX3005P real-time PCR machine (La Jolla, CA) using protocol that was previously described by Aly et al. (2008).

Clinical and Post-Mortem Examination
All quails were apparently healthy. Post-mortem examination did not indicate any abnormalities except adult helminthes in the intestine and the ceca.

Occurrence of most Pathogenic Bacteria in Oral and Cloaca Swabs
As illustrated in Table 1   Results showed that the prevalence S. aureus isolates was 19.1%. 6.4% of the S. aureusisolates was coagulase positive strains. The lowest prevalence was Proteus spp. (7.4%) L. monocytogens (4.3%) and Klepsila (3.2%).

Bacterial Isolation from Examined Body Sites
Results tabulated in Fig. 2 revealed the percentage of bacterial species from examined body organs (liver, lung and heart). The highest rate of isolation from organs was from the heart followed by liver and lung. Examination of all isolates of E. coli spp. and salmonella spp. collected from heart by API 20 test and PCR revealed that32/35 (91.4%) were E. coli spp. and 12/13 (92.3%) were confirmed as Salmonella spp.

Resistance Pattern of E. coli and Salmonella spp. Isolates
As illustrated in Table 2, E. coli and Salmonella spp. isolated from heart blood showed high resistance to multiple drugs. Over 50% of both E. coli and Salmonella spp. isolates were found resistant against sulbactin-ampicillin, amoxicillin and claveulinic, colistin, oxytetracycline, clindamycin and gentamycin.

Direct Parasitological Examination
Chewing lice were detected in between feather of wings and body in 35/94 (37.2%) of quails. Twenty seven (28.7%) of quails harbored one or two species of nematode helminthes. The first one was Heterakis spp. which was recovered from the caeca whereas the second was Ascarida galli spp. which was recovered from the small intestine.

Influenza A Virus Detection and Isolation Trials
The trials for detection and isolation of influenza viruses in oral and cloaca swabs by direct detection by RT-PCR and allantoic fluids from inoculated SPF embryonated chicken eggs were found negative.

Discussion
Importance of wild birds as potential vectors of some zoonotic disease has received recent renewed empirical interest. Studying the existence of pathogens in wild birds may serve as a useful tool for examining the spread of other diseases amongst birds and from birds to others. However, information regarding the normal gastrointestinal bacterial flora is limited for the majority of wild bird species, with the few well-studied examples concentrating on bacteria that are zoonotic and/or related to avian species of commercial interest.
Migratory bird's play an important role in transmission of the diseases because of their great mobility from area to another which plays a role as potential vectors for disease may regard domestic animals and human health (Abulreesh et al., 2007). Quails pass through seashore of Sinai during autumn migration from Central Europe. They arrive at the shore exhausted and easily caught in hunters' nets because they fly very low as they reach shore. Many of the residents of that areas hunt these birds easily and either eat or sell it out in the public markets. In the present study, results revealed that despite none of tested quails showed characteristic clinical symptoms or pathological lesions, a high occurrence of some zoonotic bacterial species were detected among them indicating asymptomatic or carrier infections. These results are inconsistent with previous studies (Leveque et al., 2003;Benskin et al., 2009;Hamad et al., 2012). This finding could be explained for many reasons. First, the migratory quails can carry bacteria for several weeks without presenting any clinical signs. Second, it has been proven that the high ability of domestic quails to acquire infection more than in migratory quail (Medani et al., 2002). Finally, the migratory quails that capable of travelling long distance start its flight from Europe, the diseased one might not be able to share in journey or die without reaching the transient area in Egypt.
The Sampling techniques for detecting incidence of some pathogenic bacteria are a major factor affecting detection rate of the pathogens. Isolation rate was differing according to the sampling sites. Our results revealed that higher isolation rate of bacteria species was in cloaca swabs in compare with oral swabs. In addition, among the internal organs, isolation rate from heart was much higher than that from lung and liver. These results were supported by previous study of Roy et al. (2012).
E. coli spp. and Salmonella spp. are the most potential pathogens causing food poisoning and posing a zoonotic hazard. In this study, the presence of E. coli was the predominant pathogen reached 37.2% of the examined quails. A nearly similar finding was previously recorded by Awadallah et al. (2013) who described a prevalence of (48%) in samples collected from wild birds in Egypt. A percentage of (38%) were reported by Rogers (2006) in California, USA. However, lower E. coli infection rates of (1-18.7%) was recorded in free living birds (Brittingham et al., 1988). The variation in prevalence rates may be attributed to the localities and bird feeding habits. Salmonella spp. Isolation rate reached (21.6%) which was much higher than that was obtained by other studies (Vlahovic et al., 2004;Hedawy and El-Shorbagy, 2007). Compared with other studies, higher solation rate of Salmonella spp. in the current study may be attributed to the fact that wild birds may consume polluted food and water. This was previously supported by Fricker (1984) who stated that waste disposal sites increases the frequency of dispersion of enteric bacteria in migratory birds.
In the current study, S. typhimurium was confirmed in 3 out of 13 (23.07%) S. typhimurium is the most serotype commonly associated with wild birds (Benskin et al., 2009). Wild quails could be considered as true reservoirs in transmission of zoonotic E. coli and Salmonella due to their indirect contact with human habitations (Ahmed et al., 2011). Therefore, the potential role of migratory quails in transmission of infectious diseases should be considered.
In this study, the incidence S. aureus was 19.1%. A similar result was agreed with a previous study (Hamad et al., 2012). Our results indicated that the migratory quails harbored some bacteria which might play a significant role in the epidemiology of some enteric bacterial pathogens. Similar results have been mentioned by Effat and Moursi (2005).
Detection of bacterial entero-pathogens in wild birds has applied through traditional microbiological techniques (Benskin et al., 2009). Our isolation was done through traditional microbiological techniques. E. coli and Salmonella spp. Isolates from heart blood were further examined by API 20 test and PCR. Results indicated that API and PCR identifications were more rapid and accurate diagnostic techniques matched with traditional techniques.
In poultry, antimicrobial agents are often continuously supplied as antimicrobial growth promoters and this has resulted in increased antibiotic selection pressure for resistant bacteria, resulting in their fecal flora containing a relatively high proportion of resistant bacteria. The use of antibiotics is supposed to be the most important factor promoting the emergence, selection and dissemination of antibiotic-resistant microorganisms in both veterinary and human medicine. Our results regarding the antimicrobial resistance pattern of E. coli and Salmonella isolates were in agreement with Roy et al. (2006) and Salehi and Reza (2010) who found that the antimicrobial resistance pattern was 50% or more of that isolates were multi-drug resistant against sulbactin-ampicillin, amoxicillin and claveulinic, colistin, oxtetracycline, clindamycin and gentamycin. However, it disagreed with that obtained by Hedawy and Wassel (2005). High sensitivity was showed in 80% of E. coli isolates was to nitrofuranation, enerofloxacine, trimethoprim,-sulphamethaxole, ciprofloxacin and ofloxacine. This finding was consistent with the findings of Blanco et al. (1997). In fact, contamination of foods with antibiotic resistant bacteria could be a major threat to public health Van et al. (2008). The diffusion of zoonotic bacteria resistant to antibiotics is an important concern for the treatment of human infections, because it can compromise the effectiveness of the therapy (Kilonzo-Nthenge et al., 2008). The increase in awareness about zoonotic infections could provide a better plan for the prevention and treatment.
Our results revealed a high incidence of one ectoparasite (chewing lice) which was detected in between feather of both wings. This finding was similar to that detected by Nursel (2010) who detect chewing lice spp. in wild quails during autumn migration. In addition, two nematode parasite species were identified. The first was identified as Heterakis spp. which was similar the finding recorded by Alan et al. (1979). In addition, Heterakis spp. was well documented in relation to blackhead in quail and/or other gallinaceous birds. The second was Ascardia gali which was recovered from the small intestine. This finding was agreed with the results of Williams et al. (2000) who found helminthes parasites in 28% of bobwhite quails.
Influenza viruses of several subtypes have been isolated from quails in North America, Europe, Asia and Africa through periodic surveillance and sporadic outbreaks. Quails could be intermediate host of IAVs Bahl et al. (2013). Wild quail may provide an optimal environment for the adaptation of wild bird AIV that could generate novel variants that can cross the species barrier to domestic poultry and human beings in spite of negative isolation trails against AIVs. Our data showed that AIVs were not detected in either oralorcloaca swabs of all quails. Similar results were clarified by Ferro et al. (2012) who mentioned that no virus was isolated from any of the suspicious samples tested. This finding might be attributed to short term of sample collection (autumn season). Therefore, the awareness to migratory birds as reservoir hosts for zoonotic pathogens should be raised.

Conclusion
Wild quail can be considered as potential vectors of zoonotic pathogens, including antimicrobial-resistant variants, which can be transferred to humans through direct contact or the food chain. Sensitivity of E. coli and Salmonella spp., strains to enrofloxacine, nitrofuranation which considered the drug of choice in case of infections. Migratory quails could serve to disperse bacteria between widely separated locations during migration. The opportunities exist for new health problems in wildlife populations emerge as well as new reservoirs of zoonotic disease to form. This work is basis of continuing efforts to periodical screening for pathogen from wild quail to predict future disease risks for wildlife and humans. Stimulating awareness to hunters, handlers and consumer about zoonotic pathogens is an important measure.

Conflict of Interests
The authors declare no conflict of interests with respect to the research, authorship and/or publication of this paper and has no direct financial relation with the commercial identities mentioned in this paper.