DETECTION OF SALMONELLA AND HELICOBACTER SPP. IN CAPTIVE WILD FELIDS

The current study aimed to investigate the presence of Salmonella and Helicobacter species in captive wild felids in addition to perform serotyping, antibiotic sensitivity test to the isolated Salmonella spp. and detection of antibiotic resistance and virulence genes. A total of 60 fecal samples were collected from 30 captive wild felids from Giza zoo and private zoo in Egypt. All animals were apparent healthy except eight African lions ( Panthera Leo ) have a history of vomiting. Samples were examined bacteriologically for the presence of Salmonella spp., followed by biochemical and serological tests. Moreover, obtained isolates were subjected to antimicrobial sensitivity testing and detection of antibiotic resistance and virulence genes. Fecal samples from lions with history of vomiting, were subjected to direct molecular identification for detection of Helicobacter spp. Overall, Salmonella spp. were isolated from 3 wild cats ( Felis chaus) . Two serovars of Salmonella were detected; S. Bovismorbificans and S. Southampton while Helicobacter felis was isolated from one African lion. Isolates of Salmonella spp. showed complete resistance to cefaclor (100%), cefoxitin (100%), and cefadroxil (100%); and very high resistance to tobramycin (66.7%), while it completely sensitive to Azithromycin (100%), Sulfa/trimethoprim (100%), Nitrofurantoin (100%), Doxycycline (100%), Amoxicillin-Clavulanic acid (100%), Fosfomycin (100%) and Oxytetracycline (100%). bla TEM and bla SHV were confirmed in Salmonella isolates showing resistant to Cefaclor and Cefoxitin, and aadA2 in S. Bovismorbificans that showing resistant to tobramycin. S. Southampton and S. Bovismorbificans have invA, stn, sopB , and hilA genes while S. Bovismorbificans carry also pefA gene as a virulence genes.


INTRODUCTION
Wild felids are strict carnivorous occupy the top of the food chain and considered as the most famous predator animals (Wang et al., 2012). They have an ecological role in regulating prey populations size and shaping animal communities (Sarasola, 2016). Furthermore, providing food for other species like scavengers, detritivores animals, and microorganisms (Marker, 2002). So, they play a critical role as a keystone species for animal community structure, function, distribution, population dynamics, and affecting behavior of interacting specie (Hopcraft et al., 2010). Loss of apex predators has negative impact on terrestrial ecosystems resulting in destabilization of herbivore-plant interactions, reduction of diversity, and loss of flexibility within ecosystems (Loveridge et al., 2016). Also in zoo and circus, it is considered as the most popular species that attract visitors and good source of income (Ripple et al., 2014).
In captivity, wild felids are susceptible to many bacterial diseases as Helicobacter gastritis, Salmonella, E. coli, Collinsella, Shigella, Proteus and Fusobacterium; however, it may be considered as a source of disease not only for other animals but also for human such as veterinarian, workers, and visitors. (Daszak et al., 2001).
Salmonella species have been isolated from the digestive contents of birds and mammals, and are capable of infecting a wide range of domestic and wild animal species (Rubini et al., 2016). In some animals, it is assumed to be an opportunistic pathogen or potentially a component of the natural gut microbiota (Dróżdż 2021) and it is considered as one of the most serious zoonotic pathogen causing several outbreaks in human around the world (Brandwagt et al., 2018, Gilsdorf et al., 2005and Stafford et al., 2002. Helicobacter infection in captive wild felids like cheetah is associated with progressive gastritis which result in vomiting, weight loss, and failure to thrive. While in human, the infection is usually linked to gastrointestinal problems, cancer, and the immunocompromised persons (Heilmann and Borchard 1991).
The current study aimed to investigate the presence of Salmonella and Helicobacter spp. in captive wild felids in addition to perform serotyping, antibiotic sensitivity test, and detection of antibiotic resistance and virulence genes for the isolated Salmonella spp.

Sampling:
The current study was performed on 30 animals belonged to family Felidae, including (16 African lions; Panthera Leo, 2 Bengal tiger; Panthera Tigris Tigris and 2 Cheetah; Acinonyx jubatus) from Giza Zoo and (5 African lions; Panthera Leo and 5 wild cats; Felis chaus) from private zoo in Egypt. A total of 60 fecal samples were collected, 2 samples from each animals; one during summer and the other during winter. Each animal was housed in separate enclosure. All animals were apparent healthy and showing no signs of diseases except eight lions in Giza zoo had a previous history of vomiting.

Collection of fecal samples:
Fresh fecal samples were collected aseptically from the floor of the animal's enclosure by removing the superficial layer of the feces and a cotton swab was inserted in the core of feces then put the swab in tube contain 10 ml peptone water.

Bacterial isolation and identification for Salmonella
Tubes of peptone water containing the fecal samples were incubated at 37º C for 18 hours before being plated onto Rappaport Vassiliadis (Himedia) broth and incubated at 42°C for 24 hours for Salmonella enrichment. A loop full from Rappaport Vassiliadis broth was streaked onto Xylose Lysine Deoxycholate media (Himedia), Hektoen enteric agar (LabM) and Salmonella-Shigella media (Himedia) then the inoculated plates were incubated at 37º C for 18-24 hours. Purification was done on the above-mentioned media tell obtaining separate, clear, and pure colonies for studying the cultural characters. (Gelaw et al., 2018) Biochemical identification of bacterial isolates was carried out by using oxidase, citrate utilization, urease, indole, methyl red, H2S on TSI, lysine decarboxylase, Dglucose-acid and gas production and Voges-Proskaure test. (Bullock and Aslanzadeh, 2013) Serological identification of Salmonella spp. A single pure colony of isolated bacteria was picked up into TSI slant. Three confirmed isolates of Salmonella were subjected to serological identification in Animal health research institute, Dokki, Giza, Egypt. The recovered Salmonella isolates were serotyped based on their polyvalent and monovalent (O) to detect somatic antigen, and polyvalent and monovalent (H) antisera to detect phase one and two flagellar antigen.

Antimicrobial sensitivity of Salmonella
The disk diffusion method was used to test antibiotic susceptibility of isolates on Mueller-Hinton agar (LabM) using 15 different antibiotic discs (Table 1). Into 5 mL of Mueller-Hinton broth (LabM), a pure colonies from 24 hours old culture were inoculated and incubated for 4-5 h until the turbidity was observed. Then, the bacterial suspension was adjusted to a density equivalent to 0.5 McFarland standard. The surfaces of Mueller-Hinton agar plates was streaked with a sterile cotton swab containing the bacterial suspension, and the plates were left for 30 min at room temperature. Then, by using an antibiotic dispenser and sterile forceps, the antibiotic discs were placed on the surface of the plate (Hudzicki 2009

RESULTS
Based on cultural characteristics and biochemical reactions, Salmonella was isolated from wild cat (Felis chaus) while the other species of wild felids appeared free from Salmonella infection. Three wild cat from total five were infected with prevalence rate 60%.
The detection of virulence genes in isolated Salmonella revealed that, the three isolates have inva, stn, sopB, and hilA genes and two isolates have pefA gene while integron gene was not detected. (Table 3

DISCUSSION
Wild animals act as a reservoir of many infectious and zoonotic diseases. Diseases transmit to people when animals are in close contact with human, such close contact can occur in zoo and captive breeding centers (Green et al., 2020).
In the present study, the rate of isolated Salmonella from wild felids were (5%) which is lower than Clyde et al. (1997) who isolated Salmonella at percent (95%) from (leopard, snow leopard, cougars, serval and caracal) and Venter et al. (2003) who isolated Salmonella at percent of (39.5%) from lions and cheetahs. This difference may returns to the type and quality of diet fed to animals in each study as the food contamination may consider as a major source for Salmonella affection where Harrison et al. (2006) isolated Salmonella at percent 28% from carcass meat fed to zoo carnivores.
From all examined felids in the current study, Salmonella was isolated only from wild cat (60%) (Felis chaus) and that may return to their diet, where the wild cat is the only species in this study fed on poultry meat while the other species fed on donkey and beef meat. Poultry meat is considered as the most common source of transmission of Salmonella (Percival and Williams, 2014) or the transmission may occur via workers due to the zoonotic nature of isolated serovars.
Salmonellosis is among the most frequent zoonotic infections in many countries and serovar Bovismorbificans is the primary cause of Salmonella outbreak infection in human in Netherlands (Brandwagt et al., 2018), Germany (Gilsdorf et al., 2005) and in Queensland (Stafford et al., 2002) S. Bovismorbificans was previously isolated from pet cats (Van Immerseel et al., 2004) and captive reptiles (Pedersen et al., 2009) while little knowledge about the isolation of S. Southampton and S. Bovismorbificans from wild felidae is available.
In the present study, Helicobacter spp. was isolated from one lion of total 8 lions (12.5%) which had a previous history of vomiting. Helicobacter spp. was previously isolated from domestic and feral cat (Ghil et al., 2009) and from wild felids like cheetah (Terio et al., 2005), lynx (Mörner et al., 2008), and in Bengal tiger (Tegtmeyer et al., 2013). Helicobacter felis has zoonotic importance and is considered as a potential pathogen in humans (Heilmann and Borchard, 1991). The fecal oral route is the main route of transmission in both cat and human (Ghil et al., 2009). The fecal contamination for water and soil play a role in spreading of the infection (Hopkins et al., 1993). The source of the infection for Helicobacter in the current study was unknown, may from the feral cat which roaming inside the zoo or from the zoo keepers due to the zoonotic nature of the isolated sample.
Bacterial adaptation to antibiotics has been extremely increased and causes significant medical problems in the last decade. Apex predators are at the top of the food chain and occupy higher trophic level. Trophic accumulation of Pollutants, and toxins can occur, in such way, the antimicrobial resistance can follow this trophic accumulation pattern from low to high trophic level (Jobbins and Alexander, 2015).
Salmonella isolates in the current study were sensitive to Sulfa/trimethoprim, Amoxicillin-Clavulanic acid, Gentamycin and Oxytetracycline, this agree with the Van Immerseel et al. (2004) In this study, blaTEM gene was detected in three isolates of Salmonella, this agree with Van Immerseel et al. (2004) who found blaTEM gene in Salmonella isolated in cat. Also, blaSHV and aadA2 antibiotic resistant genes were detected in three isolates of Salmonella.
The severity of disease caused by genus Salmonella depends on virulence genes such as invA gene which enables it to invade, penetrate and cause infection in host epithelial cells (Mubita et al., 2020). Such essential gene was recorded in three isolates of genus Salmonella from wild cats in the current study.
There are many virulence genes in Salmonella responsible for pathogenicity such as sop gene that encode Salmonella outer proteins and the hilA gene, these genes are important for penetration of cells and survival of Salmonella in macrophage (Ammar et al., 2016). All of these gene were recorded in all Salmonella isolates in the current study, this agree with Van Immerseel et al. (2004) who detected SopB gene in all Salmonella isolates in his study and with Pathmanathan et al. (2003) who found hilA gene in Salmonella bovismorbificans.

CONCLUSION:
Data in the current study highlight on the zoonotic nature of Salmonella and Helicobacter spp. which may infect the captive wild felids and cause a hazard for veterinarians and zoo keepers which routinely dealing with these animals in addition to detection for the antibiotic resistance and virulence genes that may pose a risk for failure of treatment of the captive wild felids.
Further studies are recommended to determine the source of infection for both zoonotic Salmonella and Helicobacter species in the captive wild felids.