Affected animals
In this study 39.10 % of donkeys have wounds. Donkeys in Omdurman and Khartoum North were similar in working age (p=0.7). But there was a significant association between area and type of work with (p=0.01).
Primary biochemical test
One hundred and twenty-two samples from wound secretions were investigated bacteriologically. Bacteria were isolated from 118 samples and 169 isolates were recovered with primary biochemical tests. Twenty three (23) isolates were gram-positive, non-motile, non-sporulated, not hemolytic, catalase-positive, oxidase-positive, oxidation fermentation test negative and aerobically growing.
Polymerase chain reaction (PCR)
The Universal primer of 16S rRNA amplified a product of approximately 550 bp for 10 isolates (15b, 46b, 56a, 69a, 124B, 211, 225, 103B, 151B and 4a). The product fragments were sequenced. A search of homology in the Gene Bank database by BLASTrelevled no results.
Whole-genome sequencing and Genomic features of the strain DaniaSudan
The genome sequence of strainDaniaSudanconsisted of 2.333.512 bp with a 38.1% GC. The final assembly (GCA-003627575.1) contained 75.967 contigs with N50 of 175 bp length. The largest coting assembled was 469.287 bp lengths.
The number of predicted coding sequences (CDS), number of contiges with (PCES), number of subsystems and number of RNAs were 2473, 353, 250 and 58 respectively. One CRISPR was idenfied.
Genomic Announcement
The whole genome sequence was send to the center for genomic epidemiology for multi-locus sequence typing (MLST) which identified sevenparticularnovel allele:ack−6, cpn60, fdh, pta−1,purA, sar−13and tuf.8 (Table2). A phylogenetic tree based on MLST showed relations to strainKM0211a (Fig. 1) Plasmid rep22 was showed in contig 23 by PlasmidFindet-2.0 Server (Table 3).Number of component sequences (WGS or clone) were 353.
Then the sequence was sent to RAST for annotation(Fig. 2)The result of RAST including many resistance genes methicillin resistant PBP2 (mecA, mecI and mec RI), TatR family (tet 38) and ANT (4’)-Ib(Table4). The organism has 31 virulence factors of disease and defense.
Annotation was added by the NCBI Prokaryotic Genome AnnotationPipeline (released 2013). https://www.ncbi.nlm.nih.gov/genome/annotation_prok/
Table 2
Allelic profile of strain DaniaSudan as determined by Multi-Locus Sequence Typing-2.0 Server
Locus
|
Identity
|
Coverage
|
Alignment Length
|
Allele Length
|
Gaps
|
Allele
|
Ack
|
100
|
100
|
400
|
400
|
0
|
ack−6
|
cpn60
|
98.6207
|
100
|
290
|
290
|
0
|
cpn60−16
|
Fdh
|
98.6667
|
100
|
450
|
450
|
0
|
fdh−10
|
Pta
|
98.4091
|
100
|
440
|
440
|
0
|
pta−1
|
purA
|
99.5
|
100
|
400
|
400
|
0
|
purA−2
|
Sar
|
99
|
100
|
300
|
300
|
0
|
sar−13
|
Tuf
|
99.3684
|
100
|
475
|
475
|
0
|
tuf−8
|
Notes: alleles with less than 100% identity found |
cpn60: Novel allele, ST may indicate the nearest ST.
tuf: Novel allele, ST may indicate the nearest ST.
fdh: Novel allele, ST may indicate the nearest ST.
sar: Novel allele, ST may indicate the nearest ST.
pta: Novel allele, ST may indicate the nearest ST.
purA: Novel allele, ST may indicate the nearest ST.
Table 3
PlasmidFinder-2.0 Server. Results
Plasmid
|
rep22
|
rep22
|
Identity
|
99.84%
|
99.84%
|
Contig
|
dan−contig21
|
dan−contig21
|
Note
|
repB(pAMalpha)
|
repB(Pvib110)
|
Accession number
|
AF503772
|
X03408
|
Table 4
Resistance genes of strain DaniaSudan identified by RAST program
Contig
|
Best Hit ARO
|
AMR gene family
|
Identity
|
1−56
|
Tet(38)
|
Major facilitator superfamily (MFS) antibiotic efflux pump
|
99.33%
|
1−112
|
mecI
|
Methicillin resistant PBP2
|
99.19%
|
1−114
|
mecA
|
Methicillin resistant PBP2
|
99.7%
|
1−113
|
mecR1
|
Methicillin resistant PBP2
|
100%
|
4−1
|
ANT(4’)-Ib
|
ANT(4’)
|
99.6%
|
Sequence data access
The whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number RBVL00000000. Bio sample SAMN10132107 and Bio project PRJNA493211.
Phylogenetic analysis of strain DaniaSudan nucleotide
By Tree Viewer 1.17.5 showed a relationship between the novel strain and M. caseolyticus subsp. hominis subsp. nov. (type strain CCM 7927T = DSM 103682T). (Fig. 3)
The prevalence of strain DaniaSudan
After the isolate 124B was identified as M.casueolyticusstrain DaniaSudan, the 10 bands were alignment with ref. sequence by BLAST program. Isolate 4a, 15b, 56b, 69a, 46b, 211 and 225 were identical to isolate 124B, shown in (Fig. 4)So we have 7 identical isolates to strain DaniaSudan. The prevalence of the strain was 4.73%, with(62.5%) of the isolaltes collected in winter, (75%) collected from the back of the animal (Table 5).
Table 5
M. caseolyticusstrain DaniaSudan isolates according to animal site, age, study area and season
Isolate
|
Age
|
Season of collection
|
Location of wound
|
Study area
|
Duration of work
|
65b
|
>10
|
Winter
|
Back
|
Omdurman
|
>8hor
|
124B
|
<10
|
Summer
|
Back
|
Omdurman
|
<8hor
|
69a
|
<10
|
Winter
|
Back
|
Omdurman
|
>8hor
|
15b
|
<10
|
Winter
|
Abdominal
|
Omdurman
|
>8hor
|
211
|
<10
|
Raining
|
Back
|
Khartoum north
|
>8hor
|
4a
|
>10
|
Winter
|
Head (eye)
|
Khartoum north
|
>8hor
|
46b
|
>10
|
Winter
|
Back
|
Omdurman
|
>8hor
|
225
|
>10
|
Raining
|
Back
|
Omdurman
|
>8hor
|
Sensitivity test
Eight identified isolates were subjected to antibiotic sensitivity test Results were shown in (Table 6). The organism was found resistant to ciprofloxacin, ceftazidime, erythromycin, oxacillin, clindamycin and kanamycin. However, the organism was susceptible to imipenem, ampicillin, cefoxitin, trimethoprim/sulphamethoxazole, cephalothin, vancomycin, neomycin, tertracycline and novobiocin and intermediate to penicillinG and chloramphenicol.
Table 6
Sensitivity test of M. caseolyticusstrain DaniaSudan to different antibacterial drugs
Antibacterial drugs
|
Result
|
Ciprofloxacin 5mcg (CIP5)
|
Resistance
|
Ceflazidime 30mcg (CAZ30)
|
Resistance
|
Imipenem 19mcg (IPM10)
|
Susceptible
|
Erythromycin 15mcg (E15)
|
Resistance
|
Ampicillin 10mcg (AM10)
|
Susceptible
|
Oxacillin 1mcg (OX1)
|
Resistance
|
PenicillinG 1U (P1)
|
Intermediate
|
Cefoxitin 30mcg (FOX30)
|
Susceptible
|
Clindamycin 2mcg
|
Resistance
|
Trimethoprim/sulphamethoxazole (1.25/ 23.76)mcg (SXT25)
|
Susceptible
|
Cephalothin 30mcg (KF30)
|
Susceptible
|
Vancomycin 30mcg (VA30)
|
Susceptible
|
Gentamicin 10mcg (CN10)
|
Susceptible
|
Neomycin 30mcg (N30)
|
Susceptible
|
Kanamycin 30mcg (K30)
|
Resistance
|
Tetracycline 30mcg (TE30)
|
Susceptible
|
Chloramphenicol 30mcg (C30)
|
Intermediate
|
Novobiocin 20mcg (NV30)
|
Susceptible
|
Clinical Signs
Clinical signs and temperatures of seven different groups were observed and recorded for 7 days. Mice in the control group didn’t show any clinical signs during the 7days.
Changes in mice temperature
The temperatures of all mice under test were measured before bacterial injected. The control group (G7) remained with no change in the temperature during the experimental period. The temperatures of (G1, G2, G3, G4, G5 and G6) were increased; the mean temperature was 40.90C. The highest temperatures were recorded in day five.
Behavioral responses of mice
The mice were observed as, Fatigue, allergic, lossind hair and having wounds in G1, G2, G3, G4, G5 and G6 see (Table7).
Table 7
Statistical association between different parameters
Parameter 1
|
Parameter 2
|
P. value
|
Dose
|
Swelling
|
0.001
|
Dose
|
developing of allergy
|
0.000
|
Dose
|
developing of wounds
|
0.025
|
Dose
|
loss of hair
|
0.005
|
Dose
|
pathogenically change in the eye
|
0.204
|
location of injection
|
swelling
|
0.001
|
location of injection
|
allergy
|
0.05
|
location of injection
|
developing of wounds
|
0.019
|
location of injection
|
Loss of hair
|
0.005
|
location of injection
|
pathogenically change in the eye
|
0.204
|
Gross Lesions
No pathogenic lesions were seen in the control group (G7), however G1, G2, G3, G4, G5 and G6 have different pathologic lesions (Fig. 5).
Microscopic Lesions
Internal organs showed pathological changes including liver, lung, kidney and spleen A, B, C, D, E and F. Moreover skin and muscle were showing pathogenic changesH, I, G, K and L respectively. The results were shown in Fig,6-9.