STUDY OF DIAGNOSIS, ASSOCIATED RISK FACTORS AND IN-VITRO ANTIBIOTIC SUSCEPTIBILITY OF SALMONELLA ISOLATED FROM OSTRICH ( STRUTHIO CAMELUS )

The ostrich meat is considered healthy as it contains low cholesterol and fat levels. Foodborne diseases caused by the bacterium Salmonella are a significant public health concern. The study was conducted to determine the prevalence, the risk factors involved, and antimicrobial susceptibility patterns of Salmonella isolates recovered from captive ostrich feces in the district of Lahore, Pakistan. There is not a single research on this issue in Pakistan up to date in ostrich. A total of 100 ostrich fecal samples were collected-and specifically cultured on Salmonella Shigella agar. The antimicrobial susceptibility was checked for different antibiotics throughthe Kirby-Bauer disc diffusion method. The 21% fecal samples were found positive for Salmonella . The disease was found to besignificantly(p<0.05) associated with risk factors; water type(p=0.027), bird’s capacity(p=0.042), cage hygiene(p=0.024), age of bird(p=0.041) andother bird species around (p=0.031) while enclosure size and diet were found non-significant. The bacterium was found highly susceptible to Amoxicillin followed by Chloramphenicol, Gentamicin, Cefotaxime, andEnrofloxacin respectively.Further Salmonella species-level diagnostic studies along with susceptibility checks of more therapeutic agents are suggested to future researchers.


INTRODUCTION
The ostrich plays a significant role in livestock businesses in underdeveloped countries, raising ostriches is a profitable way to earn foreign currency by selling meat and skins (Abbas et al., 2018;Abbas et al., 2018b;Fujiharaet al., 2004).Ostriches have been identified as a potential bacterial reservoir.Salmonellosis is an infectious zoonotic disease caused by Salmonella spp.and is one of the most serious problems for humans, animals, and birds (Abbas et al., 2018c;Oludairo etal., 2013).It can be transferred to humans by the consumption of contaminated food products obtained from food-producing animals.The declining productivity, higher mortality, and the associated risk of chicken products for the safety of human food, enteric illnesses are major concerns of salmonellosis in the poultry industry (Rabsch et al., 2001;Collard et al., 2008;Ramya et al., 2012).A significant public health concern is caused by the approximately 2600 serovars of Salmonella (Namata et al., 2009).
In ostrich chicks,Salmonellais the major cause of mortality.Commercial ostrich farming began in Pakistan in 2012 as an alternative to livestock and grain production.Ostrich leather, meat, eggs, feathers, and other by-products are in high demand all over the world (Ambreen et al., 2021).To develop ways to lower the risk of human infection, it is essential to identify and characterizeSalmonella in ostriches and their surroundings.The risk factors linked to ostriches contracting salmonella infection have been the subject of several investigations.For instance, South African research on ostrich farms discovered that the incidence of Salmonella was greater.This finding suggests that overcrowding may be a risk factor for Salmonella infection.The use of antibiotics in ostrich feed was cited in another Iranian study as a risk factor for the emergence of Salmonella strains that are resistant to antibiotics.Poor hygiene and sanitation procedures, insufficient ventilation, and the presence of infected wildlife or other animals close to the farm are some other possible risk factors for Salmonella infection in ostriches.The risk of Salmonella transmission can also rise when birds are moved between farms, especially if they are not thoroughly checked for infection before being introduced to a new flock.The diseases caused by Salmonella require antibiotic therapy.Salmonella and other foodborne diseases are of extreme concernfor the rise of antimicrobial resistance in animals, birds, and humans.The World Health Organization has highlighted the rise of antibiotic-resistant non-typhoid Salmonella strains (Cha et al., 2013).
According to our knowledge, there is not a single research study on this issue in Pakistan yet.The objective of the current study was to determine the prevalence of Salmonella in ostriches being kept for either commercial or domestic or display purposes in district Lahore along with the risk factors associated with the occurrence of disease and to check the susceptibility of the selected drugs against Salmonella in ostriches.

MATERIALS AND METHODS
Sampling Strategy: A total of 100 fecal samples from ostrich having diarrhea, weight loss, and anorexia were collected from public and private ostrich rearing sites in district Lahore by usinga convenient sampling method.The fecal samples were collected either directly from the rectum of ostriches or freshly passed feces.The fecal samples were placed in a sterile container.The samples were transported in a cooler/box containing ice packs immediately to the Postgraduate Laboratory, Department of Veterinary Medicine, UVAS Lahore forfurther processing.
Bacteriological examination: Pre-enrichment of the fecal sample was done using buffer peptone water (1g in 250 ml buffer peptone water) then incubated overnight at 37 o C.After incubation 0.1ml of buffer peptone water was inoculated on 9ml tetrathionatebroth(selective enrichment broth) and then incubated at 42 o C for 24 hours.A loopful from each selective enrichment broth (after incubation) was streaked onto the following selective agar media Salmonella shigella agar by streak plate method to observe the colony morphology of Salmonella.Salmonella isolates were further confirmed by Gram staining and biochemical Tests (Indole Red Test, Methyl Red Test, VogesProskauer, and Citrate Utilization Test (IMVIC).

Risk factors analysis:
Data regarding risk factors; animal age (adult, sub-adults, chicks), diet (balanced, not balanced), water for drinking (filtered, tab, pond), enclosure type (small, medium, large), Bird capacity (not crowded, over-crowded), Cage hygiene (good, poor) and Bird species around (yes, no) was collected through questionnaire filled on spot while sample collection from different farms.

Assessment
of antimicrobial susceptibility: Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion technique using Mueller-Hinton agar according tothe Clinical Laboratory Standards guidelines (CLSI) manual (Rincón-Gamboa et al., 2021).The antimicrobial agents tested and their corresponding concentrations were as follows: Amoxicillin (AMX: 30 mu/g), Enrofloxacin (ENF: 5 μ/g), Gentamycin (GM: 10 μ/g), Cefotaxime (CTX: 30 μ/g) and Chloramphenicol (CH: 30 μ/g).After incubating the inoculated plate overnight at 37 o C the susceptibility of the Salmonella to each antimicrobial agent wasmeasured and the results were interpreted followingthe CLSI manual.When the minimuminhibitory concentration (MIC) of the Salmonella isolates for a given antimicrobial comes in the intermediate to sensitivity range, Salmonellawas considered to be not resistant against that antimicrobial agent.

Statistical Analysis:
The risk factors were analyzed by using Chi-square (χ2) and thedata on antibiotic susceptibility was analyzedthrough descriptive statistics.The whole data analysis was carried out through SPSS version 21.0.

Diagnosis of Salmonella:
On Salmonella Shigella agar, opaque, translucent colorless, smooth round colonies with a black center appeared as shown in Figure 1.While examination of the stained microscopic slide revealed rod-shaped gram-negative bacteria as shown inFigure 2. Prevalence ofSalmonella: Out of 100 samples,21%were found confirmed positivefor Salmonella on selective media.Among the total positive, following clinical signs with percentages of ostrich were observed;anorexic (57.14%), suffering fromdiarrhea (80.95%),weight loss (38.09%),depressed(85.71%)andhistory of retarded growth(28.57%).

Risk factors associated with the occurrence of Salmonella in ostrich :
Theanalysis of risk factors revealed that out of 7 variables, 5 were identified as potential risk factors (p<0.05) for Salmonella in ostriches (Table 1).These variables werewater source, bird's capacity, cage hygiene condition, access of other species to farm, and age.While enclosure type and diet type were non-significantly associated risk factors.

Antimicrobial susceptibility:
The susceptibility of all antibiotics for isolated salmonella was determined as shown in Figure 3.After biochemical confirmation, 21 isolates of Salmonella were used for antibiotic sensitivity pattern and the results are shown in Table 2 and Figure 4.
The study showed that Salmonella has the highest susceptibility toward Chloramphenicol.Salmonella showed the highest resistance towards Amoxicillin and Enrofloxacin.

DISCUSSION
Salmonellosis is a food-borne and zoonotic disease infecting a wide range of vertebrate hosts.When humans come into close contact with the bacterium from wild animals or ingest meat products, they might become infected with Salmonella.Because there is a lack of knowledge about prevalent Salmonella serotypes, monitoring of farms is essential to controlling and preventing the spread of Salmonella to people, particularly in the case of the ostrich business (Oludairo et al., 2013).In this study 21% percentage positivity of Salmonellawas observed.This finding was almost in agreement with (Hameed et al.,2019) who isolated Salmonella from ostriches and recorded a prevalence of 28.57%.Despite disagreeing with (Metawea, 2013) who discovered Salmonella in an ostrich farm with an incidence of 8.6%.According to MA et al., 2016 Salmonella were present in 20.8% and 24.4% of all samples collected from chick yards and adult yards, respectively.Oliveira et al., 2006 discovered that none of the 80 samples of ostrich droppings from different age groups collected in the southeast Brazilian region had Salmonella.In the Ismailia, it was discovered that Salmonella was present in faces, cloacal swabs, and internal organs of ostrich flocks older than three months, but not in flocks younger than two months (Effata et al., 2003).In the intestines and caeca of ostriches; more Salmonella spp.were identified with overall percentage positivity 18.5% (46/248) (Vanhooser et al., 1995).According to research by Gaedirelwe and Sebunya, 2008 in Botswana;Salmonella was isolated from 51.6% (16/31) of the ostriches.The difference in Salmonella spp.prevalence may be due to variation in housing type, hygienic condition, and species susceptibility variations.Variation in the percentage positivity of ostrich for salmonellosis may be due to variations in climatic and rearing conditions of each geographical area.
In this investigation, we discovered 6 percent, 5 percent,and 10 percent percentage positivity of Salmonella in ostriches offered filtered, pond, and tab water respectively.Comparatively to the 36% observed in commercial poultry drinking water in Thailand, the isolation rate from drinking water was 21% (Sasipreeyajan et al.,1996).The reason is that Salmonella infections can be caused by contaminated drinking water, which has also been linked to the spread and persistence of the disease in poultry farms (Adzitey et al., 2011).Nonetheless, our study's isolation rates were also somewhat higher than the reported 3.3% (poultry water) isolation rates from layer farms in Northern India (Singh et al.,2013).It was found that higher percentage of positivity (19%) in crowded flocksas compared to less crowded flocks (2%).The reason is that more number of bird per area lead to maintenance of infection as less hygienic conditions and more chances of transmission between birds (Khalilzadeh et al., 2023).According to this study small cage size, less cage hygiene, more bird's species around, less balanced diet, and younger age were responsible for higher prevalence 13%, 14%, 12%, 15%, and 11% respectively.According to another study Ostriches that were clinically and subclinically infected with Salmonella were found to be in fence-to-fence contact with other animal species (Brassó et al., 2023).Small cage size leads to overcrowding and causes stress to birds which is responsible for less immunity and higher prevalence in the case of small cage size.Poor hygienic condition and higher exposure to otherbird species leads to the sustainability of infection in the environment for a long duration and are responsible for widespread transmission to other nearby rearing facilities so responsible for high prevalence.At younger ages less immune development is the reason for higher prevalence in younger age.
In the farming of birds; rodents are a significant source of Salmonella infection (Carrique-Mas et al., 2009).According to the research, the introduction of flocks infected with Salmonella may cause transmission in rodents, which becomes a source of the microbe for subsequent flocks and creates a challenging challenge (de Freitas Neto et al.,2009).The differences in frequency between farms may be explained by the sanitary practices used in each farm, the housing arrangement, the water source, the location of the sample, the season, the addition of antibiotics, and the health of the flock.The antibiotic susceptibility of the Salmonella strains was assessed in this investigation.All the isolates were sensitive to Amoxicillin,Chloramphenicol, and Gentamicin.Resistance to Cefotaxime and Enrofloxacin was present.According to Mohammadzadeh et al., 2017 all the isolates were resistant to ampicillin and tetracycline.According to research by Shetty et al., (2012) and Murugkar et al., (2005), ceftriaxone and ciprofloxacin were the most effective antibacterial medications.According to another research, it was identified that Salmonellais resistant to Tetracycline, Penicillin, Chloramphenicol, Erythromycin, Amoxicillin, Gentamicin, Ceftriaxone, Nalidixic Acid, Levofloxacin, and Vancomycin, and susceptible to Azithromycin (Jahan et al., 2017).This variation can result from the varied antibacterial medications that were utilized in various areas.

Conclusion:
This study concluded that the prevalence of Salmonella in ostriches in district Lahore is 21%.The water source, bird capacity in pens, pen hygiene, different bird species present around the farm, and age of the bird are the significantly associated risk factors for the occurrence of salmonellosis in ostriches.Salmonella is highly sensitive to chloramphenicol so we can use it to treat its infection in ostriches.

Recommendations:
The current study furnished the baseline data but further Salmonella species-level diagnostic studies along with susceptibility checks of more therapeutic agents are suggested for future researchers.There is also a need to study this organism in ostriches on the molecular level (molecular characterization, study on immunogenic and pathogenic genes, and work on its vaccine development due to both veterinary and public health concerns.

Figure 1 :
Figure 1:Appearance of Salmonella as small, round, translucent, and colorless colonies with a black center on Salmonella Shigella agar.

Figure 2 :
Figure 2:Salmonella bacteria are rod-shaped (bacilli) and appear as Gram-negative cells under Gram's staining