ISRAEL JOURNAL OF
VETERINARY MEDICINE
IN VITRO AND IN VIVO PATHOGENICITY
STUDIES OF ESCHERICHIA COLI ISOLATED FROM
POULTRY IN NIGERIA.
Vol. 58 (1) 2003
M.A. Raji1, J.O. Adekeye1, J.K.P. Kwaga2 and J.O.O. Bale3
1. Dept. of Veterinary Pathology and Microbiology,
2. Dept. of Veterinary Public Health and Preventive Medicine and
3. Dept. of Animal Reproduction, National Animal Production Research
Institute Shika, Ahmadu Bello University Zaria-Nigeria.
Abstract
Diseases of poultry caused by Escherichia coli are of economic importance
in the poultry industry in Nigeria. The organism is responsible for 30-40%
mortality in broiler industry and the losses do not take into consideration weight
loss and poor carcass quality when birds are infected with E. coli.
The in-vitro pathogenicity was also conducted using Congo red, motility
and hemolytic tests while the in vivo pathogenicity study was conducted using
day-old-chicks. The results showed that 40% of the isolates from clinical cases
were Congo red positive while 56% and 39% of the isolates from Simtu farm
and NAPRI were Congo red positive respectively. The results of hemolysis
indicated that none of the isolates from Simtu farm was hemolytic while only 2
(10%) out of 20 clinical colibacillosis isolates were hemolytic and 1 (2%) out of
50 isolates N.A.P.R.I showed a zone of hemolysis on 5% sheep blood agar.
Most of the E. coli isolates were motile and all the clinical cases and NAPRI
isolates.
In vivo pathogenicity of all the serotyped isolates were compared using
day-old-chicks inoculated with various isolates. Strain differences in the
response of the birds were noted in the mortality. Clinical cases, 91-2000
(O9:K30), 98-2000 (O8:K50) and 92-2000 (O9:K30) produced clinical signs of
severe lameness, depression, diarrhoea, and loss of weight and 100% mortality
in in vivo pathogenicity studies in day-old chicks.
This study highlights the serogroups of E. coli in this environment, and is
also the first documentation of E. coli serotypes in Zaria-Northern Nigeria and
successfully demonstrated the pathogenicity of these isolates for day old chicks.
Introduction
Escherichia coli is a major pathogen of worldwide importance in
commercially raised poultry, contributing significantly to economic losses in
both turkeys and chickens. E. coli has been associated with a variety of diseases
in birds, including enteritis, arthritis, omphalitis, coligranuloma, septicemia,
salpingitis, and complicated air sacculitis (1). Its role in chronic respiratory
diseases in meat-type chickens is well documented (2), and its pathogenicity has
been correlated with numerous extrinsic and intrinsic bird-related factors and
conditions. The extrinsic factors include environment, exposure to other
infectious agents, virulence and levels and duration of exposure. Intrinsic factors
affecting susceptibility include age, route of exposure, active and passive
immune status and breed and strain of chicken (3). Microbial characteristics
associated with virulent avian E. coli include antibiotic resistance (4) production
of colicins and siderophores (5), type 1 pili (6), plasmids (7), motility (8)
hemolytic reaction (9) and embryo lethality (10). Resistance to the lytic action of
host complement has also been implicated as a virulence-associated parameter in
E. coli isolates from chickens and domestic animals with colisepticemia (10)
Most of the serotypes isolated from poultry are pathogenic only for birds, but a
few are also associated with disease conditions of other animals (11). The
purpose of the present study was to examine the virulence of some serotypes of
Escheichia coli isolated from poultry using day old chick lethality and to
correlate it with other virulence factors associated with pathogenic avian E. coli.
Materials and Methods
A total of ninety-three E. coli isolates from clinical cases of colibacillosis
and dead -in-shell embryos were subjected to Congo red, hemolysis and motility
tests. Twenty of them were from clinical cases of colibacillosis and twenty-eight
from Simtu farm and forty-five isolates from N .A P.R.I farm were from dead-inshell embryos.
A) Serotyping Assay method
Serotyping of the isolates was done by standard slide agglutination tests with
antisera against somatic antigen groups at South Africa Laboratory by Dr.
Maryke Henton, of Department of Bacteriology, Onderstepoort Veterinary
Institute according to standard methods described by Orskov et al. (12) and
Glantz et al. (13).
B) Virulence Assay
a) Haemolysis assay
E. coli isolates were propagated on blood agar base supplemented with 5%
washed sheep erythrocytes. Blood agar plates then incubated at 37 OC for 24 hrs
and colonies producing clear zones of haemolysis were then recorded as
hemolysin positive (14).
b) Motility (using SIM Agar) test.
The medium was prepared and inoculated with each isolate of E. coli and
incubated at 37 OC for 24hrs after which motile bacterial was seen to spread
from point of inoculation into the agar as a paint brush (8, 14).
c) Fermentation of carbohydrates.
E. coli isolates were characterized by their ability to utilize maltose, lactose,
sucrose, dulcitol, adonitol, salicin, raffinose, dextrins, xylose, rhamnose and
mannitol. Bromothymol blue broth base was prepared and used as colour
indicator. Each isolate of E. coli was inoculated into prepared sugar and
incubated at 37OC for 24hrs. The positive test turned from blue to yellow, while
the negative reaction remained blue (7, 15).
d) Congo red Binding Assay.
The medium used for determination of Congo red binding of the isolates was
prepared according to the formula of Berkholl and Vinal (15). Trypticase Soy
Agar was supplemented with 0.003% Congo red dye (Sigma) and 0.15% bile
salts (Coast Louis Missouri). Each isolate was cultured on a separate plate and
incubated at 37 OC for 24hrs. After 24hrs incubation, the cultures were left at
room temperature for 48hrs to facilitate annotation of results (15). Invasive E.
coli were identified by their ability to take up Congo red dye. The positive
isolates produced red colonies. The negative isolates appeared as colourless.
e) In Vivo Virulence Test using Day Old chicks
Twenty-four isolates of E. coli serotyped from clinical cases and dead —inshell embryos were used. One hundred and twenty day- old cockerels were
obtained from NAPRI hatchery. The birds were fed with chick mash (Feed
MastersR) and water provided ad libitium.
All the tested strains were grown and prepared according to the method of
Chulasiri and Suthienkul (4). The McFarland standard turbidity of 3X108 was
diluted in Brain Heart infusion broth in one to tenfold dilution factor and last tube
of 107 was plated on MacConkey agar and incubated at 37OC for 24hours. The
colony forming units (CFU/ml) was determined by standard plate count method
(17). The birds were assigned into experimental groups of five. Birds were
injected subcutaneously with 0.2ml of brain heart infusion broth containing
106or 107 CFU per isolate. The actual dose was determined by viable cell counts
of the inocula. Birds were maintained for seven days post inoculation and
monitored daily for mortality. Birds that died before day 7 and those that
survived till end of the experiment were necropsied to determine the gross
pathological lesions on their organs and cultural examination was also done on
liver and pericardium.
Results
The result of haemolysis indicated that none of the isolate from Simtu farm
was haemolytic, while only 2(10%) 20 of clinical colibacillosis isolates were
haemolytic and 1(2%) 50 isolates N.A.P.R.I showed zone of haemolysis on 5%
sheep blood agar. Most of the E. coli isolatesd from clinical cases and dead-inshell embryos were not haemolytic. The result of Congo red assay showed that
55% of clinical cases of colibacillosis were Congo red positive, while 50% and
42.2% of E. coli isolates from Simtu farm and N.A.P.R.I farms respectively were
positive for Congo red (Table 1). Congo red uptake was demonstrated in 3(12 of
out pathogenic isolates) while the remaining pathogenic isolates were Congo red
negative. There was no correlation with Congo red dye from this finding.
The commonest serogroups in the clinical cases were O8:K50 (2) and
O8:K30 (2) followed by O86:K62 (1) while the remaining fifteen of the isolates
belong to rough strains. The serotypes from dead-in-shell embryos from Simtu
farm were O8:K50 (2), O4:K3 (1), O13:K11 (1) and O78:K80 and O8:K41 were
recovered from a single isolate, S195. The rough isolates were demonstrated in
twenty of the isolates while two isolates were not included in the group sent to
South Africa for serotyping and one of the isolate got broken in transit. The
results of serotypes from National Animal Production Research Institute were as
follow. The serogroups O9:K34 (3) O9:K9 (2), while the remaining serotypes
O8:K50, O26:K60, O8: K-, O99: K, O137:K79, O9:K28, O112:K68 occurred
singly (Table 2).
The result of the pathogenicity in day-old chicks indicated that 100%
mortality was recorded in the clinical cases of colibacillosis 91-2000(O9:K30),
92-2000(O9:K30) and 98-2001(O8:K50), while 20% mortality were produced by
38-2001(O8:K50) and 40-2001(Rough strain) isolates. The isolate 30-2001
(O86:K62) was not pathogenic for day-old chicks. The results of the Simtu farm
isolate showed that only isolate S68 (Rough) produced 100% mortality in dayold chicks and only one isolate S195 (O78:K80 and O8:K41) produced 80%
mortality in day-old chicks and two isolates belonging to number S26 (O4:K3)
and S420 (O13:K11) produced 40% mortality in day-old chicks. The results of
pathogenicity studied from NAPRI farm isolates showed that 100% mortality
was produced with the isolate number N16 (O8: K?) and N25 (O99: K). While
80% mortality was produced with only one isolate N6 (O9: K). Four of the
isolates produced moderate mortality (60% mortality) in day—old chicks
belonged to number N23 (O9:K34), N14 (O9:K28) N58 (O9:K34) and N28
(O8:K50). One of the isolate produced 40% mortality in day-old chicks belonged
to N53 (O112:K68). Two of the isolates produced 20% mortality were N19
(O9:K9) and N60 (O26:K60), while isolates number N36 (O9:K34) and N36
(O137:K79) were not pathogenic for day old chicks. The inoculum size used in
the experiment varies from 106 - 107 as previously described by Cloud et al.
(7).
The clinical signs presented from sick birds before they died were vents
pasted with faeces, depression, lameness, anorexia, and loss of weight, ruffled
feathers and weakness.
The post-mortem findings indicated congested and enlarged liver, heart, and
spleen. The gall bladder was also distended with bile. The lungs were also
congested. The cultures from liver and pericardium also were positive both for
dead birds and sick birds that survived till the end of the experiment.
Pathogenicity was classified as highly virulent if the mortality was from 60100% and twelve of the isolate actually belonged to this group. The moderate
group was those with mortality from 20-40% and three of the isolates were in this
group. The non-pathogenic group was those with mortality of 20% and less nine
isolates belonged to this group (Table 3).
A large percentage of all isolates showed enhanced activity in the
fermentation of rhamnose (100%) and lactose (100%). While fermentation of
ducitol, mannitol, xylose showed (99%) each for both pathogenic and nonpathogenic isolates. Comparatively, the fermentation of adonitol by the large
number of non-pathogenic serotypes especially six of the low virulence
serotypes. Metabolic activity was similar among the different isolates and for
most substrates and could not be associated with pathogenicity (Table 4). An
exception was bile esculin where 100% of the non-pathogenic isolates
hydrolyzed this salt.
The most pathogenic serotypes in this study were O9:K30, O8: K, O99: K,
and rough strains showed 100% mortality in day-old chicks.
Dose response (Table 4) shows the dose related response of day-old cockerel
chickens to the E. coli isolates did not vary considerably even when a lower dose
(106) of pathogenic strains were inoculated. Lesions and mortality were
produced in the day-old chicks. At least 107 CFU/ ml of highly pathogenic E.
coli was required to consistently produce lesions and /or mortality in more than
50% of the inoculates. The weak pathogens failed to kill the birds regardless of
dose.
Discussion
Two isolates from clinical cases 10%(2/20) and one from the National
Animal Production Research Institute (N.A.P.R.I) 2.25%(1/45) were hemolytic
E. coli. Hemolytic strains are frequently isolated in pure culture from the
intestine and mesenteric lymph node of pigs dying from oedema disease (36) and
a frequent association of hemolytic strain of Escherichia coli with gastro-enteritis
and septicemia in calves and humans hase also been documented (27). The nonhemolytic strains of were responsible for colibacillosis and dead-in-shell
embryos (7, 19). This study also agrees with previous workers that nonhemolytic E. coli were associated with colibacillosis and dead-in-shell embryos
in Nigeria.
The results of the Congo red binding assay indicates that 55% of cases of
colibacillosis produced Congo red positive while 50% and 42.2% from Simtu and
NAPRI farms were Congo red positive, respectively. The pathogenicity of the
Congo red positive isolates was not correlated with Congo red uptake. A number
of workers have also reported that Congo red binding did not correlate well with
pathogenicity (20). Previous study also indicated that isolates of virulent avian E.
coli can be identified by their ability to bind Congo red (CR). The characteristic
of CR binding constitutes a moderately stable, reproducible, and easily
distinguishable phenotypic marker. The stability of the CR phenotype is greater
in some isolates than in others. The loss of CR binding parallels the loss of
virulence for chickens and mice (15). Berhkof and Vinal (15) reported that
generally, most CR-positive E. coli isolates will lose the ability to bind CR if
sub-cultured on complex media (such as blood agar). No consistent correlation
between sugar fermentation, Congo red, bile esculin, motility, serotypes and in
vivo pathogenicity for day-old chicks was found in this study. It was also
observed in a study (7), that E. coli isolates from colisepticemia produced
inconsistent results on correlation with sugar fermentation test, motility and in
vivo pathogenicity test for day-old chicks.
In this present study all the pathogenic and non-pathogenic isolates for dayold chicks actually fermented ducitol which disagreed with the finding of Baeul
(16), who reported that higher percentage of pathogenic than apathogenic E. coli
strains were ducitol positive and salicin negative in their studied. Some
researchers have also noted that these serogroups share similar biochemical
patterns, such as the fermentation of certain sugars or enzymatic reactions. It has
been proposed that high metabolic activity might be characteristic of highly
virulent strains or specific serogroups associated with colibacillosis (7,14) and
dead-in-shell embryos. (17). In vitro pathogenicity test using Congo red could
not be clearly correlated with pathogenicity. The majority of Congo red negative
E. coli were pathogenic for day-old chicks, while only very few Congo red
positives were pathogenic for day-old chicks. This finding disagreed with the
earlier studies by Berkhoff and Vinal (15). This may be due to the fact that most
of the isolates studied were of serogroups O1, O2, and O78. The possible
explanation was given by Spears et al. (21) that a large plasmid in Shigella
flexneri encoded virulence and Congo red binding were at closely associated, but
separate, loci. They concluded that the loci were separate and that other nonvirulence-related loci such as those encoding cell wall and other outer-membrane
synthesis could affect Congo red binding phenotype with virulence was not
absolute. The low positive correlation between the Congo red tests and other tests
reflects the low degree of direct relationship between the diagnostic tests (Congo
red uptake and embryo lethality).
This is the first report of E. coli serotyping from Northern Nigeria. Of a total
of eighty-six isolates from colibacillosis and dead-in-shell embryos serotyped, 5
isolates belonged to O8:K50. This was followed by serotypes O9:K34 (3 isolates)
while 2 isolates each belonged to serotypes O9:K30 and O9:K9. The remaining
serotypes occurred singly O8: K, O78:K80, O8:K41, O13:K11, O137:K79,
O112:K68, O26:K60, O4:K3, and O9: K28.
The findings in this study disagree with the reports of (7,19), where high
incidences of serotypes O1, O2 and O78 were reported in cases of colibacillosis
and dead- in-shell embryos. In this study, serotypes O8, then O9 and O78 in that
order were most frequently encountered in this work. Hinton and Linton (22) had
reported the association of colibacillosis with O8 and O9 in South Africa. The
very low frequency of isolation of some serotypes known world wide as
pathogenic for avian in this environment needs to be investigated further, by
studying a higher number of isolates to be able to make any definitive
conclusions or converting the rough into smooth isolates for easy serotyping.
This study has however given additional evidence of the presence of some E. coli
serotypes in this environment and further confirmed that strains of similar
serotypes can be isolated from different countries. Ninety-five isolates in this
investigation typically represented the species. Eighty-five of these strains were
serologically typed in detail in South Africa. Strains belonging to serogroups O8
were isolated most frequently from poultry infections followed by O9, and O78.
In this investigation, it is significant to note that both O8 and O9 have been
isolated by the Nigerian workers (17, 24). Tekdek, (23) in Zaria reported that the
commonest serogroups identified in calves were O8, O26, O15, O75 and O6. Of
all the E. coli serotyped only one was a pathogenic strain O78:K80 that by
experimental infections caused enteric colibacillosis in lambs. In his report, the
commonest ‘O’ serogroup observed was O8. This serogroup also occurred most
frequently in poultry in this study in the same environment and there is likelihood
that there could be cross-infection from lambs to poultry. Falade (24), in Oyo
State-Nigeria isolated O141 and O139 serogroups, which are not among the
serogroups normally associated with pathogenic lesions in poultry.
O8 serogroup isolated in this study have been associated with hatchery
losses and early chick mortality in India (25). This appears to be first
documentation of several serogroups of E. coli associated with dead-in-shell
embryos and clinical cases of colibacillosis in Zaria and environs.
There were no untypable strains in the present study. Orajaka and Mohan
(17) found only 26% untypable strains from dead-in-shell embryos. Very little
information is available on the association of untypable E. coli strains with
embryonic mortality. However, Rosenberger et al. (31) noted that O2 serotypes
and untypable avian E. coli are among the virulent avian E. coli. This observation
was not confirmed in this study.
Pathogenicity of E. coli isolates from clinical cases of colibacillosis from
Avian Unit of Veterinary Teaching Hospital, A.B.U-Zaria, and dead-in-shell
embryos from NAPRI was done by inoculating a standardized amount of E. coli
subcutaneously into of susceptible day-old cockerel chicks from Simtu Farms.
Based on mortality and lesions that occurred during a 7-day post-inoculation
period, isolates were characterized as being high, intermediate, and a virulent in
pathogenicity. Birds were susceptible to subcutaneous E. coli administration; and
confirms report by other researchers (11,35,30). In a study by Sojka and
Carnagham (11), mortality was consistently reproduced in birds following
subcutaneous injection with E. coli serotypes especially with O1, O2, O78, O73
and O08. In studies by Sojka and Carnagham, (11) and by Kabilika and Sharma
(32), both failed to produce mortality in day-old chicks with the following
serotypes O9:K28, O141: K85 and O8:K?.
In the present study, however, the serotypes 09:K30 (two isolate), 08:K (one
isolate) 099:K (one isolates) 08:K50 (one isolate) and one rough produced 100%
mortality in day-old chicks, and serotypes O8:K50 (one isolate), O86:K62 (one),
O9:K34 (one) O137:K79 (one) were not pathogenic for day-old chicks. There
was variation in the response of the birds to inoculation of the serotyps. The
mortality and lesions produced from inoculation of day-old chicks were almost
the same in all pathogenic E. coli. The typical clinical signs of colibacillosis were
noticed in the experimentally inoculated birds. These were vents pasted with
feces, lameness, roughed feathers, and loss of weight, anorexia, and weakness.
The postmortem findings were enlarged and congested liver, lungs, and heart.
The gall bladder was distended with bile and the intestine was filled with watery
feces. The spleen was also enlarged and congested. This agreed with the finding
of Grosheva and Rackhmaniana, (33). The amount of inoculum given to each
bird varied from106 to 107. The pathology and mortality with these inoculum
sizes were unrelated to pathogenicity while lower numbers of some of the strains
also produced the same mortality and lesions.
Serogroups O9:K30 and O8: K were most frequently isolated and were quite
pathogenic suggesting these are emerging pathogens of some significance in
NAPRI-produced chickens. Serogroup O78:K80 was isolated only once and
appeared to be important as indicated by other researchers (3,11). The rough
strains from clinical cases and Simtu dead-in- shell embryos strains were also
assessed. The rough strains from clinical cases showed 100% mortality for dayold chick whereas the rough strain from Simtu produced 20% mortality. There is
need to assess the remaining rough strains of all E. coli isolates before a
conclusive statement could be made. Notably Ciosk reported that transformation
of smooth (S) to rough (R) forms was accompanied by a decrease in virulence in
all his experiments (34).
LINKS TO OTHER ARTICLES IN THIS ISSUE
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1.
TABLES
Table 1: In vitro pathogenicity studies on E. coli isolates from dead-in-shell embryos
and clinical cases of colibacillosis.
Pathogenicity Test
Clinical cases of
Colibacillosis
Dead-in-shell
Embryos-Simtu
Farm
0/28(0 %)
Dead-in- shell
Embryos-NAPRI
Farm
1/ 45(2.22%)
Haemolysis
2/20(10%)
Congo Red
11/20(55%)
14/28(50%)
19/45(42.2%)
20/20(100%)
27/28(99.8%)
45/45(100%)
dye
Motility
Table 2: E. coli Serotypes isolated from dead-in-shell embryos and clinical cases
of colibacillosis
Serotype
Clinical cases of
Colibacillosis
Dead-in-shell
EmbryosSimtu Farm
Dead-in- shell
EmbryosNAPRI Farm
Total
08:K50
2
2
1
5
09:K30
2
-
-
2
086:K62
1
-
-
1
09:K9
-
-
2
2
09:K28
-
-
1
1
09:K34
-
-
3
3
099:K?
-
-
1
1
08:K?
-
-
1
1
04:K3
-
1
-
1
026:K60
-
-
1
1
0112:K68
-
-
1
1
0137:K79
-
-
1
1
013:K11
-
1
-
1
078:K80
-
1
-
1
Rough
14
20
32
66
Not typed
1
3
1
5
Total
20
28
45
93
08:K41
Table 3: In vivo pathogenicity studies in day-old chicks.
S/no
Laboratory
CFU
Mortality
1
N38
3.52X107
0/5
2
N36
7.70
X107
3
S109
4
%
Mortality
Serotype
Virulence
No of
birds
with
lesions
No of
birds
without
lesions
-
O137:K79
None
-
5
0/5
-
O9:K34
None
-
5
7.25
X107
0/5
-
O8:K50
None
-
5
N60
4.56
X107
1/5
20
O26:K60
None
3
2
5
S420
3.90X107
2/5
40
O13:K11
Moderate
3
2
6
38-2001
5.52X107
1/5
20
O8:K50
None
2
3
7
98-2001
9.84X107
5/5
100
O8:K50
High
5
-
8
N58
7.04X107
3/5
60
O9:K34
High
4
1
9
N16
3.97X107
5/5
100
O8: K-
High
5
-
10
N53
5.86X107
2/5
40
O112:K68
Moderate
3
2
11
N25
6.53X107
5/5
100
O99: K-
High
5
-
12
S26
9.82X107
2/5
40
O4:K3
Moderate
3
2
13
91-2000
7.2X106
5/5
100
O9:K30
High
5
-
14
S195
5.0X106
4/5
80
O78:K80
High
4
1
O8:K41
15
92-2000
6.3X106
5/5
100
O9:K30
High
5
-
16
S68
1.38X107
5/5
100
Rough
High
5
-
17
40-2001
2.08X107
1/5
20
Rough
None
2
3
18
N23
1.44X107
3/5
60
O9:K34
High
4
1
19
N6
4.90X107
4/5
80
O9:K9
High
4
1
20
N14
2.50X107
3/5
60
O9:K28
High
3
2
21
N19
1.62X107
1/5
20
O9:K9
None
2
3
22
N28
1.92X107
3/5
60
O8:K50
High
3
2
23
S69
3.06X107
1/5
20
O8:K50
None
2
3
24
30-2001
7.36
X107
0/5
-
O86:K62
None
-
5
Pathogenicity classification
Highly virulent=50-100% (N=12)
Moderate or intermediate=20-40% (N=3)
Avirulent =20% or less (N=9)
Table 4: Correlation between sugar fermentation, bile esculin hydrolysis, Congo red
assay, hemolysis, motility, serotype, and in vivo pathogenicity in day-old chicks.
Serotype
O137:K79 +
+
-
+
-
+
+
+
+
+
-
+
-
O9:K34
+
+
-
+
-
+
+
+
-
-
+
+
-
O8:K50
+
+
+
+
+
+
+
+
+
+
+
+
+
O26:K60
+
+
+
+
-
+
+
+
+
-
+
+
-
O13:K11
+
+
+
-
-
+
+
-
+
-
+
+
-
O8:K50
+
+
+
-
-
+
+
+
+
+
+
-
-
O8:K50
+
+
+
-
-
+
+
-
+
-
+
-
-
O9:K34
+
+
+
+
-
+
+
+
+
+
+
+
-
O8:K?
+
+
+
+
-
+
+
-
+
+
+
+
-
O112:K68 +
+
+
+
-
+
+
+
+
+
+
+
+
O9:K?
+
+
+
+
-
+
+
+
+
+
+
+
+
O4:K3
+
+
+
+
-
+
+
+
+
+
+
-
-
O9:K30
+
+
+
+
-
+
+
+
+
+
+
+
-
O78:K80
+
+
+
+
+
+
+
+
+
-
+
+
+
O9:K30
+
+
+
+
-
+
+
+
+
+
+
-
+
Rough
+
+
+
+
-
+
+
+
+
-
+
+
-
Rough
+
+
+
+
+
+
+
+
+
+
+
+
-
O9:K34
+
+
+
+
+
+
+
+
+
-
+
+
-
O99:K9
+
+
-
+
+
+
+
-
-
+
+
+
+
O9:K28
+
+
-
+
+
+
+
-
+
+
+
+
-
O9:K9
+
+
+
+
-
+
+
+
+
-
+
+
-
O8:K50
+
+
-
+
-
+
+
+
-
+
+
+
-
O8:K41
O8:K50
+
+
+
+
+
+
+
+
+
-
+
-
-
O86:K62
+
+
+
+
-
+
+
-
+
+
+
+
-