Abdalla Aerobic bacteria isolated from internal lesions of camels at Tambool slaughterhouse

Objective: This cross-sectional study was conducted from January to June 2013 to estimate the prevalence of internal lesions in slaughtered camels at Tambool slaughterhouse and to identify the aerobic bacteria colonizing these lesions. Material and methods: A total of 280 carcasses of camels were thoroughly inspected during post-mortem examination and samples were collected from internal lesions and cultured onto different culture media. Isolated bacteria had undergone testing of several biochemical activities for species identification and susceptibility to certain antibiotics. Results: Internal lesions were detected in 25.0% (95% CI of ±5.07) of the carcasses and were observed in the lungs, liver and heart. Statistical association analyses showed that origin, sex, breed, and body condition were related to detection of lesions. A number of 179 isolates of aerobic bacteria belonging to 9 genera were grown and included Staphylococcus species (32.0%), Escherichia coli (26.0%), Streptococcus species (18.0%), Corynebacterium species (5.0%), and Bacillus species, Salmonella species, Pseudomonus species, and Klebsiella species; each of them 4.0%, and Shigella species (2%). Drug susceptibility testing showed that chloramphenicol was the most effective antibiotic against Staphylococcus species, Corynebacterium species, and Bacillus species, while penicillin g 10 was more effective against Streptococcus species. Ciprofloxacin 5 μg and ampicllin/sulpactum 20 μg were effective against E. coli, Salmonella species, Klebsiella species, Shigella species, and Pseudomonas species. Conclusion: It can be concluded that internal lesions are prevalent in camels slaughtered at Tambool slaughterhouse and many species of bacteria were colonizing these lesions, hence, evaluating the economic magnitude of internal lesions resulting in partial and/or total condemnation of organs is warranted.


INTRODUCTION
Africa hosts the biggest camel population in comparison to other continents, with the Sudan ranking second to Somalia in terms of camel population density (Keskes et al., 2013).The country has nearly 4.5 million camel heads that are mainly found in the so-called camel belt in the arid and semi-arid zones and are primarily raised for riding, baggage carrying, pulling, and for hides, meat and milk and for social purposes (Tigani et al., 2007;Faye et al., 2011).
Like other animals, camels can be infected by many infectious diseases caused by different species of bacteria, viruses, parasites and fungi (µgrane and Higgins, 1985; Alhendi, 2000).These disease can be categorized by the infected body system or organ, e.g.digestive system diseases, cutaneous infections, respiratory diseases, and mastitis, in addition to metabolic disorders (Alhendi, 2000).Also, these diseases have a seasonal trend and associated with place (Agab and Abass, 1998).In 2004, peste des petits ruminants (PPR), a viral disease known to cause gastro-intestinal and respiratory symptoms associated with morbidities and mortalities in small ruminants, emerged in camels in the Sudan as has been reported by Khalafalla et al (2010).During the outbreak, PPR caused diarrhea, colic, respiratory distress, abortions, and sudden death of apparently healthy animals.PPR has also been recently reported in camels in Iran (Zakian et al., 2016).Another example for emerging disease in camels is the Middle East respiratory syndrome coronavirus (MERS-CoV) which was first reported in 2012 (Sharif-Yakan and Kanj, 2014).
A lesion is a damage through an injury or a disease, such as a wound, ulcer, or abscess.When it occurs in an internal organ or tissue, it's called internal lesion.Staphylococci, Pasterullae, Escherichia coli and Corynebacterium species were isolated and identified from camels' internal lesions and abscesses (Younan et al., 2005;Zubair et al., 2004;Aljameel et al., 2014).Internal lesions are economically important due to the subsequent partial or total condemnation of affected organs (Younan et al., 2005).This study was carried out to determine the prevalence of internal lesions in slaughtered camels at Tambool slaughterhouse, isolate and identify bacteria from these lesions, and to determine the antimicrobial susceptibility profile of the bacteria isolated from the lesions.

Study area:
This study was carried out in Al-Butana plains in the central region of the Sudan at Tambool slaughterhouse, Al-Gazeera State.Tampool slaughterhouse is around 130 km away to the Southeast of the Sudan's capital town Khartoum.

Study design:
A cross-sectional study was conducted for a period of six months, from January to June 2013 at Tambool slaughterhouse.This slaughterhouse was purposively selected because this area is known to be heavily populated with camels and the habit of eating camel meat is popular, too.

Sample size calculation and sampling strategy:
The sampling size (n) was determined as per the standard formula of Thrusfield (2007), and n was calculated to be 280 animals.Moreover, animals included in the study were selected by systematic random sampling.

Post-mortem inspection:
Post-mortem inspection was performed visually, by palpation and in situ slicing of certain lymph nodes (LN) in the head, thorax, and abdomen, and organs like lungs, liver, the heart and others following the procedure described by Asseged et al. (2004).
Specimen collection: Specimens were taken aseptically and preserved in sterile bags in ice and transported to the Microbiology Laboratory of the College of Veterinary Medicine, Sudan University of Science and Technology, where isolation and identification of bacteria were performed.
Laboratory procedures: Different types of culture media were used in this study together with biochemical tests reagents for primary isolation and identification of bacteria according to Barrow and Feltham (2003).

Preparation of bacteriological media:
According to the instructions of the manufacturers, 40 gm, 59 gm, and 25 gm powder bases of blood agar, MacConkey agar, and nutrient agar were, respectively, weighted, dissolved, and autoclaved at 121C/15 pound per square inch for 15 minutes.The dissolved powder bases were poured into Petri dishes, dispensed properly, and conserved at 4C in a refrigerator until used.Mannitol agar, egg yolk agar, deoxycholate citrate agar (DCA), eosin methylene blue agar (EMB), and Simmons citrate agar were prepared according to the instructions of the manufacturers as well.
Bacteriological protocol: Blood agar, MacConkey agar and nutrient agar were streaked on by swabs from the specimens and incubated at 37C for 24 h.Gram stained smears were made from grown colonies, besides to observing hemolysis patterns, colony size, color, surface and shape.Sub-cultures were made onto special media for purification of the isolates.
Identification of the isolated bacteria : Gram staining was carried out according to Barrow and Feltham (2003).The prepared slides were examined microscopically under oil emersion objective lens.Biochemical tests, as shown in Table 1 and 2, for identification of the species of the detected isolates were carried out as described by Barrow and Feltham (2003), and included oxidase, catalase and motility tests as well as Indole, Vogas Prokauer, nitrate reduction and urease tests.In addition to Lactose, Sucrose, Trehalose, Xylose, Raffinose, Maltose, Mannitol, Sorbitol, Simmons's citrate and O/F reactions and H2S production tests.

Antimicrobial susceptibility testing:
The drug susceptibility profile of the isolated and identified bacteria was tested on nutrient agar.A bacterial suspension was cultured by streaking all over the agar, then sensitivity discs were placed on top of the cultured agar.Penicillin 10, Amoxicillin 10, and Chloramphenicol efficacy was tested against Gram positive bacteria while Tetracycline 30 µg, Ciprofloxacin 5 µg, Ampicillin/Sulbactam 20 µg efficacy was tested against Gram negative bacteria.After incubation in a humidity chamber for 24 h at 37C, the clear zone surrounding the antimicrobial disc was recorded in millimeter (mm).
Statistical analyses: Appropriate statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS) version 18.0 (SPSS Inc, USA).

RESULTS
Internal lesions were detected in camels slaughtered at Tambool slaughterhouse with variations between different age groups, breeds, sexes, and origin of animal as presented in Table 3.The overall prevalence was 25.0% (n=70/280) with 95% CI of ±5.07.The prevalence of internal lesions in camels from Darfur, Rezaigy camels, she-camels, and camels with poor body condition was significantly higher than camels from other locations, breeds, male camels, and camels with good and moderate body condition.
The logistic regression analysis revealed that geographical origin (Darfour) sex (she-camels), breed (Rezaigy), and body condition (poor) were associated with odds of observing internal lesions in camels' carcasses (Table 4).Lesions were detected in different internal organs including lungs, liver and heart (Table 4).Lungs inflammation (pneumonia) (51.4%) was the most observed pathology followed by hepatic lesions (45.7%) and the least (2.90%) was inflammation of the heart muscle (myocarditis).However, no other internal organs showed any kind of pathologies during the period of the study.
As displayed in Table 8 and 9, chloramphenicol was the most effective antibiotic against Staphylococcus spp., Corynebacterium spp., and Bacillus spp., each with an inhibition zone of 10 mm, while Penicillin g 10 showed more efficacy against Streptococcus spp. with an inhibition zone of 10 mm.Ciprofloxacin 5 µg and Ampicllin/Sulpactum 20 µg were effective against E. coli, Salmonella spp., Klebsiella spp., Shigella spp., and Pseudomonas spp. with inhibition zones of 8 and 10 mm.

DISCUSSION
The camel is a hardy animal when compared to other domestic animals and is less susceptible to many of the diseases that affect other livestock species in the same ecosystem or geographical area (Awol et al., 2011).Many apparently healthy slaughtered camels were found to have one or more internal lesion in one or more organ (Bekele, 2008;Awol et al., 2011).In this study, a quarter of the examined carcasses of camels had internal lesions in the lungs, livers, and hearts and this was lower than the reports of Awol et al. (2011) and Bekele (2008) who found    The prevalence of internal lesions in carcasses of camels from Darfur was significantly higher than in carcasses of camels from other geographical areas.The same was observed for Rezaigy camels when compared with camels from other breeds.This might be due to the stress of the long-distance travel.These animals had walked for a distance of 1000 km, at least, to arrive in Tambool before being slaughtered.
The difference between the prevalences reported from males and females were significantly different.This was in contrast to the findings of Tenaw et al. (2015) who found no significant difference in the prevalence of lungs lesions between sexes.The significant difference reported herein, might likely be due to the big number of female camels investigated in comparison to the number of male camels.
Significant difference in the prevalence of internal lesions in camels with different body conditions was observed in the present study.Camels with poor body condition were showing the highest prevalence, however, camels with good body condition were showing the lowest prevalence.Tenaw et al. (2015) made the same observation and reported that the lungs of camels having poor body condition score were found to be highly affected with pneumonia when compared to camels with medium and good body condition score.The immune competence of the animal and resistance to disease are probably reduced as result of the poor body condition score and hence this situation increases the vulnerability of the animal to infections and formation of internal lesions.
Origin, sex, breed, and body condition were associated with the detection internal lesions in the univariate analysis using chi square.However, age was not significantly associated with internal lesions detection.Prevalence of lesions due to hydatid cyst in the lungs of slaughtered camels was related to body condition score (P≥0.000) while it was not influenced by sex.And, lesions due to hydatid cyst in the livers were not associated with body condition score and sex (Tenaw et al., 2015).Prevalence of emphysema and pneumonia in the lungs of slaughtered camels was not related to body condition score and sex (Tenaw et al., 2015).Cultural variations as well as social activities, animal production and improper disposal of dead animals could probably lead to noticed variation (Yifat et al., 2011;Tenaw et al., 2015).
The prevalence of lesions in the lungs herein was higher than the prevalences reported by  2015) found hepatic lesions in 3.8% to 29.0% of the livers of the slaughtered camels in Nigeria and Bangladesh.Hepatocytes destruction induced by worms like liver flukes and the metabolism of toxic materials that have been absorbed from the gut and their removal enhance formation of liver lesions and colonization of the lesions by opportunistic and pathogenic bacteria (Scanlan and Edwards, 1990).
Condemnation of hearts due to myocarditis was observed in low percentage in this study typifying the findings of Tenaw et al. (2015) who reported that 1.55%(n=6) of the hearts of slaughtered camels were found with lesions.However, no other internal organs showed any kind of pathologies during the period of the study.
A number of aerobic bacteria, Gram positive cocci and bacilli and Gram negative rods, belonging to 9 genera were detected in this study.Nuseba et al. (2014) was able to grow 81 isolates of different micro-organisms, of which 56 (76.0%) were Gram-positive bacteria, 20(24.0%)were Gram negative bacteria, and 5(7.0%) were fungi.The growth inhibition efficiency of penicillin G 10, amoxicillin 10, and chloramphenicol was tested against Gram positive bacteria and of tetracycline 30 µg, ciprofloxacin 5 µg, and ampicllin/sulpactum 20 µg against Gram negative bacteria.Chloramphenicol was the most effective antibiotic i n inhibiting the growth of Gram positive bacteria, amoxicillin 10 was fairly effective and penicillin G 10 did not inhibit the growth of Bacillus species.Ampicllin/sulpactum 20 µg was the most effective antibiotic inhibiting the growth of Gram negative bacteria, ciprofloxacin 5 µg was fairly effective and tetracycline 30 µg did not inhibit the growth of Pseudomonas species.This was similar from the findings of Tiwari et al. (2015) who found that chloramphenicol was among the highly sensitive drugs and conversely amoxycillin and ampicillin/sulbactum were resisted in most cases.Shuaib et al. (2016) found out that ciprofloxacin (5 µg), amikamicin (30 µg), cephalexin (30 µg), and impenen (10 µg) efficiently inhibited the growth of most isolated bacteria in their study while cefotaxime, ampicillin/sulbactam (30 µg), bactricin (10 µg), ceftizoxime (30 µg) and amoxacillin (30 µg) were the less effective or totally ineffective antibiotics in inhibiting bacterial growth.Cholaramphenicol (30 µg) and penicillin-G (10 unit) inhibited bacterial with varying degrees.Shuaib et al. (2016) mentioned that the dynamic of drug-resistant bacteria in animals in the Sudan is most likely related to the storing conditions of veterinary drugs, liberated uncontrolled trading, and arbitrary use of these drugs by animal owners.

CONCLUSION
It can be concluded that internal lesions are prevalent in camels slaughtered at Tambool slaughterhouse.Geographical origin, sex, breed, and body condition were significantly associated with the detection internal lesions.Many Gram positive bacteria and Gram negative bacteria were isolated from the samples collected from these lesions including Staphylococcus species, E. coli, Streptococcus species, Corynebacterium species, Bacillus species, Salmonella species, Pseudomonus species, Klebsiella species, and Shigella species.Chloramphenicol and penicillin G 10 were effective against most Gram positive bacteria and ciprofloxacin 5 µg and ampicllin/sulpactum 20 µg were effective against Gram negative bacteria.The economic magnitude of internal lesions resulting in partial and/or total condemnation of organs should be evaluated.Molecular epidemiology of the isolates needs to be investigated.

ACKNOWLEDGEMENT
The assistance provided by the technical staff of the Microbiology Laboratory of the College of Veterinary Medicine, Sudan University of Science and Technology, during carrying out this work is valued.

Table 3 :
Observed prevalences of internal lesions of camels by origin, sex, age breed, and body condition and univariate associations of risk factors with internal lesions in camels in in Tambool slaughterhouse (January to June 2013)

Table 4 :
Multivariate associations of risk factors with internal lesions in camels in in Tambool slaughterhouse (January to June 2013)

Table 5 :
Number of camel carcasses with internal lesions in lungs, livers, hearts and other organs in Tambool slaughterhouse (January to June 2013)

Table 6 :
Number of Gram positive bacteria isolated from internal lesions of lungs, livers, and hearts of camels in Tambool slaughterhouse (January to June 2013) Abubakar et al. (2010)f the camel carcasses had lesions in Ethiopia and the report ofAbubakar et al. (2010)who saw lesions in 64.0% of the carcasses in Nigeria.These observed difference in occurrence of internal lesions could be due to variation in sample size (number of examined carcasses) or due to variation among geographical areas from where the camels originated or even due to previous diseases affected the animal in the past.

Table 7 :
Number of Gram negative bacteria isolated from internal lesions of lungs, livers, and hearts of camels in Tambool slaughterhouse (January to June 2013)