https://doi.org/10.52973/rcfcv-e34307
Received: 19/08/2023 Accepted: 27/09/2023 Published: 01/01/2024
1 of 11
Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34307
ABSTRACT
Diarrhea in calves can be caused by bacteria, viruses, and parasites.
Among bacteria, Escherichia coli is considered responsible for the
appearance of enteric diarrhea and septicemia in these animals,
conditions that require immediate attention. Among E. coli infections
of calves, more focus is placed on intestinal pathogenic (InPEC)
infections, and extra – intestinal pathogenic (ExPEC) infections are
ignored. This study aims to reveal which E. coli pathotype causes
the infection as molecular and serotype and to reveal the differences
according to the age groups of the factors in the herd. Blood and
fecal samples of 10 calves aged 3 – 15 d with diarrhea were analyzed.
The primary agent causing enteritis was determined by examining
the stool samples with BoviD – 5 Ag. Then, samples were subjected
to culture and identication processes. It was determined that the
stool samples had 2/10 with E. coli K99, 4/10 with rotavirus, and 4/10
with mixed rotavirus infections and Cryptosporidium spp. E. coli was
detected from all blood samples by hemoculture. The study isolated
only the SepEC and ETEC groups from samples. All SepEC isolates
were determined to carry type 1 pilus responsible for adhesion. In
addition, it was determined that 9/10 of the SepEC group carried the
colicin V gene responsible for pathogenicity. Also, all E.coli isolated
from calves aged 3 – 15 d were found to be resistant to antibiotics. In
conclusion, primary enteritis is caused by rotavirus Cryptosporidium
spp. and ETEC. However, it was determined that SepEC group E.
coli causing septicemia showed different antigenic and genetic
features than E. coli in the intestinal tract. The virulence factors
of the SepEC group may vary due to genomic plasticity, and their
antigenic structures should be more closely examined and added
to vaccine test studies.
Key words: Escherichia coli; septicemia; calf diarrhea; antimicrobial
resistance
RESUMEN
La diarrea en los terneros puede ser causada por bacterias, virus
y parásitos. Entre las bacterias, Escherichia coli se considera
responsable de la aparición de diarrea entérica y septicemia en estos
animales, afecciones que requieren atención inmediata. El objetivo
del estudio fue denicar el patotipo causante de diarrea y septicemia
entérica y factores asociados. Se analizó muestras de sangre y heces
de 10 terneros de 3 – 15 d de edad que presentaron diarrea. Los análisis
de las heces determinaron E. coli K99 en un 2/10, rotavirus el 4/10,
rotavirus y Cryptosporidium spp. el otro 4/10. El hemocultivo registro
presencia de E. coli en el 10/10 de las muestras. Todos los aislamientos
de SepEC portarón pilus tipo 1 responsable de la adhesión, un 9/10
porto el gen de la colicina V responsable de la patogenicidad.
Además, se encontró que todas las E. coli aisladas de terneros de 3
a 15 d de edad eran resistentes a los antibióticos. SepEC causante
de septicemia mostró características antigénicas y genéticas
diferentes a las de E. coli en el tracto intestinal. En conclusión, la
enteritis primaria es causada por rotavirus, Cryptosporidium y ETEC.
Se pensó que los factores de virulencia del grupo SepEC pueden
variar debido a la plasticidad genómica y sus estructuras antigénicas
deberían examinarse más de cerca y agregarse a los estudios de
prueba de vacunas.
Palabras clave: Escherichia coli; septicemia; diarrea terneros;
resistencia antimicrobiana
Microbiological characterization and genetic analysis of bacteria isolated
from blood cultures and fecal samples in calves with symptoms of
septicemia and diarrhea
Caracterización microbiológica y análisis genético de bacterias aisladas de hemocultivos
y muestras fecales en terneros con síntomas de septicemia y diarrea
Ali Uslu* , Zafer Sayin , Asli Balevi , Aysegul Ilban , Osman Erganis
Selcuk University, Faculty of Veterinary Medicine,
Department of Microbiology. Konya, Türkiye.
*Corresponding author: aliuslu@selcuk.edu.tr
Characteristics of bacteria isolated in calves with septicemia-diarrhea symptom / Uslu et al. ______________________________________
2 of 11
INTRODUCTION
Losses due to calf diarrhea are among the largest economic losses
for dairy and meat producers [1]. It has been reported that 57 and
53% of fatal diseases in calves under one month of age are diarrhea
[2], leading to dehydration, depression, sepsis, and death [3]. Calves
born with agammaglobulinemia due to failure of passive transfer
immunity (FTPI) cause primary intestinal infection due to bacteremia
and a systemic inammatory response that causes septicemia [4].
Antimicrobial treatment should be applied in cases of diarrhea that
present with septicemia, fever, and coma [3] to avoid animals' death
due to lack of antimicrobial therapy [5].
Multiple enteric pathogens such as viruses, bacteria, and protozoa
are part of the etiology of diarrhea in calves [6], the most common
being bovine rotavirus (BRV), bovine coronavirus (BCoV), bovine viral
diarrhea virus (BVDV), Salmonella enterica, Escherichia coli, Clostridium
perfringens, and Cryptosporidium spp. [7]; E. coli infections are
examined under two pathotypes, intestinal pathogenic E. coli (InPEC)
and extra – intestinal pathogenic E. coli (ExPEC). E. coli diarrheas
(InPEC) are examined under six patho groups; enterotoxigenic
E. coli (ETEC), enterohemorrhagic/shiga toxin – producing E. coli
(STEC/EHEC), enteropathogenic E. coli (EPEC), enteroinvasive E.
coli (EIEC), enteroaggregative E. coli (EAEC) and in extra – intestinal
E. coli infections (ExPEC) [8].
Of these bacteria, ETEC is the most common cause of diarrhea
in calves. ETEC adheres to epithelial cells with mbriae antigen
(commonly known as F5) and secretes heat – labile toxin (LT) and
heat – stable toxin (ST) [9, 10]. Because the pH is less than 6.5, the
epithelial cells of the distal part of the small intestine are most
suitable for colonization by ETEC, causing villous atrophy and damage
to the lamina propria [11].
In ExPEC pathotype O6, O8, O11, O15, O20, O25, O27, O78, O128, O148,
O149, O159, O173 somatic antigens are dominant [12]. Extra – intestinal
infections are those causing septicemia, urinary tract infections, and
neonatal meningitis produced by (ExPEC) such as (SepEC) septicemia
associated E. coli, (UPEC) uropathogenic E. coli and (NMEC) neonatal
meningitis E. coli. It has been reported that this group of pathogens
has various virulence factors (such as toxins, polysaccharide capsule,
adhesin, invasin, iron acquisition factors, and lipopolysaccharides) and
genomic plasticity [13]. SepEC bacteria adhere to the mucosal surface,
colonizing it with adhesins. Following intestinal infection, the mucosal
surface is destroyed [14]. SepEC resists the antibacterial activity of
the calf's innate immune system. Thanks to the siderophores, the
bacteria can multiply in iron – limited tissues and then pass to the
circulatory system and all internal organs [15].
This study aimed to determine the relationship between bacterial,
viral, or parasitic agents that cause enteric infections. It was also
aimed to reveal the relationship between phenotypic/genotypic
characteristics between SepEC in the bloodstream and E. coli strains
in gut microbiota.
MATERIALS AND METHODS
Collection of samples
Newborn calves were monitored on the farm with a capacity of
3,500 dairy cows in Konya – Turkiye, which presented symptoms of
diarrhea and sepsis during the cold and wet seasons of December
and January. Blood and feces samples were collected, following the
methodology proposed by Fecteau [16], classifying diarrheal feces
according to color and mucus. At least 10 mL of blood was taken
directly from the jugular vein until it reached the line specied on
the aerobic blood culture bottle (BACT/ALERT® FA PLUS BMX410851).
Teng of feces from the rectums of calves with diarrhea were collected
into fecal collection containers (10022 – 168, VWR) containing peptone
water. The samples were delivered to the laboratory for analysis on
the same day via cold chain.
Evaluation of stool samples with rapid lateral ow test
The eld lateral ow test named BoviD – 5 Ag (RG13 – 02, Bionote,
USA) was used according to the kit instructions to determine the
agent causing diarrhea from diarrheal stool samples. A sample was
taken from the diarrheal stool with a swap. The swap containing
the feces was transferred to the sample tube containing the assay
diluent and was homogenized until the feces was separated from
the swap. It was waited for 30 s for the sediments to settle. And
the supernatant was taken, and 4 drops were added to each well
for diagnose (bacterial; E. coli K99, viral; rotavirus and coronavirus,
protozoal; Giardia spp. and Cryptosporidium spp.). It was waited for
10 min for the test and control lines to take shape.
Microbiological examination of blood samples (blood culture) and
agent isolation – identication
The blood culture bottle was incubated at 37
o
C 50 rpm in a
shaking incubator (MIR – 254 – PE, Panasonic) [17]. Blood cultures
were passaged at 24, 48, 72 h intervals onto blood agar (NCM0075A,
LabM), selective media MacConkey agar (MC) (70143, Sigma Aldrich),
and Eosin Methylene Blue agar (EMB) (70186, Sigma Aldrich) and petri
dish with media incubated 16 – 24 h at 37
o
C and 5% CO
2
. At the end of
incubation, lactose – positive E. coli colonies were observed as metallic
green colonies on EMB agar and pink – colored colonies on MC agar.
Microbiologic examination of stool samples and isolation and
identication of agents
For E. coli isolation, each stool sample transported to the laboratory,
one drop of feces was passaged on MC and EMB agar, which are
selective media for E. coli isolation, and incubated at 37°C for 16 – 24h.
At the end of incubation, lactose – positive E. coli colonies formed
metallic green – colored colonies on EMB agar and pink – colored
colonies on MC agar [18]. For Salmonella isolation, 1 g of stool was
passaged in 9 mL peptone water (CM1049, Lab M) for 16 – 24 h at 37°C
for pre – enrichment. Then, 1 mL of the peptone enriched sample was
transferred to new tube containing 9 mL of Rappaport Vassiliadis
Salmonella Enrichment Broth (HP007, LabM), which is a Salmonella
selective enrichment medium, and incubated at 42°C for 24 h. Then,
a loopful of the enriched medium was passaged to XLT – 4 selective
agar (CM1061, Thermo Scientic) and incubated at 37°C for 48 h and
observed whether black – colored Salmonella colonies would form [19].
Biochemical Identication of enteric Gram – negative bacteria
All bacterial isolated that grew on MC agar or were detected as
Gram – negative bacilli by Gram staining were biochemically conrmed
with a tipped tube test. The agents were evaluated in terms of
lactose – glucose – H
2
S in the rst tube, mannitol movement in the
second tube, and urea – indole in the third tube, and the bacteria were
identied according to the report by Lassen, 1975 [20].
______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34307
3 of 11
Serotyping of isolated Escherichia coli
The strains were stored at -80
o
C with the appropriate code number.
Since the ETEC group expresses mbriae antigen, all isolated E. coli
were passaged on MINCA agar (potassium dihydrogen phosphate 1.36
g·L
-1
, disodium hydrogen phosphate 8.05 g·L
-1
, casamino acids 1.0 g·L
-1
,
glucose 1.0 g·L
-1
, agar 15.0 g·L
-1
trace salt solution 1 mL).
The latex test detected E. coli isolated on MINCA agar with F5 (K99)
antiserum (51173, SSI Diagnostica). For detecting somatic antigens, E.
coli to be passaged on MINCA Agar, antiserum Pool1 EPEC/VTEC/STEC
O26, O103, O111 EAEC, O145, O157 (44292, SSI Diagnostica København,
DK), antiserum Pool2 EPEC O55, O119, O125ac, O127, O128ab (44293,
SSI Diagnostica København, DK) were conrmed by latex testing
with antiserum Pool3 EPEC O86, O114, O121, O126, O142 (77713, SSI
Diagnostica København, DK).
Also, these pool antigens could detect EAEC serotypes such as
O127 in Pool2 and O86 in Pool3. In addition, the isolated strains were
heat inactivated in an autoclave (NC100, Nuve) at 121
o
C under 1 ATM
pressure for 75 min, SAT antigens (~108·mL
-1
) were prepared, and
serotyping was performed with E. coli O1 STEC, O2 STEC, O8 ETEC,
O9, 033, O38, O78 ETEC/EAEC and O101 ETEC monoclonal antisera
(SSI Diagnostica København, DK) according to lace formation by SAT
method in microplate [21]. The strains were serotyped with a total of
23 different somatic antigens. For selective isolation of VTEC, STEC,
EHEC Group, E. coli O157:H7 was identied by passaging on sorbitol
MacConkey agar E. coli O157:H7 MUG Agar (44782, Merck) and conrmed
by latex test with antiserum pool1 (44292, SSI Diagnostica). The strains
were also passaged on blood agar containing 5% sheep blood to detect
the presence of hemolysin enzymes. Nonmotile, lactose – negative
colonies grown on EMB and MC agar were typed as EIEC. They were
also molecularly conrmed by polymerase chain reaction (PCR). E. coli
that did not t the other typing classes were molecularly typed with
aggregative features and conrmed with Pool2 and Pool3 antisera and
named EAEC. E. coli O9 K99 ATCC 31616, E. coli O157 ATCC 43895, and
non – pathogenic E. coli ATCC 25922 were positive controls.
Determination of antibiotic susceptibility of isolates
The antibiotic resistance of the isolates was determined by the disk
diffusion method. E. coli isolates were incubated at 37°C on tryptic
soy broth (1054590500, Sigma Aldrich) for 12 h. After being adjusted to
McFarland (DEN, Biosan) 0.5 standard turbidity, they were passaged
onto 100 mL of Mueller Hinton agar (70191, Sigma Aldrich), spread with
a drigalski and incubated at 37°C for 12 h [22].
Zone diameters were recorded in mm. Antibiotic resistances were
determined according to Clinical and Laboratory Standards Institute
(CLSI) 2022 data. As a result of antimicrobial testing, it is dened
as resistant (R) if the zone cap is smaller than the CLSI breaking
point ranges, intermediate (I) if it is in the range, and sensitive (S)
if it is larger or equal. If the bacterial isolate was sensitive to 3 of
the antibiotic groups, it was categorized as multidrug – resistant
(MDR); if it was sensitive to two or only one antimicrobial group, it
was categorized as extensively drug – resistant (XDR); and if it was
resistant to all of them, it was categorized as pan drug (PDR) [23].
An analysis was made to determine the most preferred antimicrobial
agents in enteric therapy in calves and their resistance to imipenem,
which is preferred in human use. These antibiotics list: P: Penicillin,
AMC: Amoxacillin, CFP: Cefoperazone, CRO: Ceftriaxone, TE:
Tetracycline, IPM: Imipenem, SXT: Trimethoprim/sulfamethoxazole,
CN: Gentamicin, ENR: Enrooxacin, E: Erythromycin. The clinical
breakpoint was based on the resistance data of CLSI veterinary
isolates. For quality control strains, E. coli ATCC 25922 were tested
with the isolates.
DNA isolation from stool samples
According to the kit instructions, deoxyribonucleic acid (DNA) was
isolated from diarrhea stools using the QIAamp DNA Stool Mini Kit
(51604, Qiagen). The quality and quantity of DNA isolates were measured
with a spectrophotometer (Nanodrop 2000, Thermo Scientic).
Identication of bacteria with molecular methods
DNA was isolated from the pre – identied agents according to the
Wizard® Genomic DNA Purication Kit protocol. The concentrations of
the isolated DNA were determined by spectrophotometer (Nanodrop
2000, Thermo Scientic). Set 1, set 2, and set 3 multiplex primers were
designed according to the study of Lee [24], and set 4 multiplex primers
were designed according to the study of Oh [25] and Vandekerchove
[26]. The primer pairs specied in TABLE I in the project were used.
For the PCR mixture, ve μL Master mix (5x), 0.1 μL forward primer
(10 pmol/ μL, 0.1 μL reverse primer (10 pmol·μL
-1
), two μl DNA (100
ng·μL
-1
) 17.9 μL sterile nuclease – free water were added for a total
volume of 25 μL. The thermal cycle (T100, Bio – Rad) was repeated 34
times with a pre – denaturation step at 94°C for 10 min, followed by
94°C denaturation for 1 min, 60°C binding for 1 min, 72°C extension
for 1 min, and nal extension at 72°C for 10 min. A 1% agarose gel
was prepared for electrophoresis (maxicell – minicell, EC Apparatus
Corporation) of PCR products. Ethidium bromide was added to the
gel to a nal concentration of 0.5 μg·mL
-1
. Gel wells were loaded with
ve μL each of PCR products and 100 bp DNA ladder. The results were
visualized by a gel imaging device (212 Pro, Gel – Logic).
RESULTS AND DISCUSSION
In the herd from which samples were collected, rotavirus was
detected in 4/10 feces with diarrhea in newborn calves. In the other
4/10, rotavirus and Cryptosporidium spp. were detected as mixed. E.
coli K99 was detected in only 2/10 (TABLE II). In all animals, the fever
was around 40°C, the hair was uffy, and the mucous membranes
were dehydrated. Only animal number 4 with rotavirus diarrhea
had a fever of 41°C (FIG. 1). It was determined that the age range of
animals infected with rotavirus and Cryptosporidium spp. was between
10 – 15 d. Animals with E. coli K99 diarrhea were aged 3 – 4 d, and this
nding is compatible with other studies [27, 28]. Salmonella spp. and
Giardia spp. were not detected in fecal samples with diarrhea. A study
conducted with 300 calves with diarrhea in the region reported that
40% of calves in the 15 – 29 – day age group had rotavirus infection,
and 12.9% had rotavirus + Cryptosporidium spp. infection [29]. In the
prevalence studies of the agents causing diarrhea in calves in Türkiye,
E. coli was detected at a rate of 9.4% – 27.45% [30, 31, 32, 33, 34].
Since studies of calf diarrhea generally focus on diarrheal agents
(bacteria, virus, or protozoa), the bacteria causing septicemia are
overlooked. However, diarrhea and septicemia calves’ prevalence
are approximately 9.26% – 31% [5, 35, 36]. In this study, no pathogens
other than E. coli were isolated from blood cultures. All the isolated
strains were conrmed to be E. coli with differential media MC and
EMB media. In addition, the results were supported by the triple tube
method. All strains did not show hemolysis on blood agar, and E. coli
Age and temperature distribution in infected calves P
Characteristics of bacteria isolated in calves with septicemia-diarrhea symptom / Uslu et al. ______________________________________
4 of 11
TABLE I
Primer sets used in the study
Bacteria Gene Primer
PCR Product
(bp)
Multiplex
Set
E. coli
ETEC F4 F – GCCTGGATGACTGGTGATTT / R – TCTGACCGTTTGCAATACCC 709 Set 1
E. coli ETEC F5 F – TTGGGCAGGCTGCTATTAGT / R – TAGCACCACCAGACCCATTT 222 Set 1
E. coli ETEC F6 F – GCGTGCATCGAAATGAGTT / R – GGTGGTTCCGATGTATGCTT 589 Set 1
E. coli VTEC F18 F – CTTTCACATTGCGTGTGGAG / R – ATTCGACGCCTTAACCTCCT 444 Set 1
E. coli ETEC F41 F – GGAGCGGGTCATATTGGTAA / R – CTGCAGAAACACCAGATCCA 941 Set 1
E. coli ETEC STa F – GAAACAACATGACGGGAGGT / R – GCACAGGCAGGATTACAACA 229 Set 2
E. coli ETEC STb F – CCTACAACGGGTGATTGACA / R – CCGTCTTGCGTTAGGACATT 480 Set 2
E. coli ETEC LT F – GGTTTCTGCGTTAGGTGGAA / R – GGGACTTCGACCTGAAATGT 605 Set 2
E. coli SHEC Stx2e F – TGGTGTCAGAGTGGGGAGAA / R – TACCTTTAGCACAATCCGCC 351 Set 2
E. coli EHEC EAST1 F – CCATCAACACAGTATATCCGA / R – GGTCGCGAGTGACGGCTTTGT 111 Set 2
E. coli EHEC mA F – TGGTGGGACCGTTCACTTTA / R – AAGGTCGCATCCGCATTAG 443 Set 3
E. coli EHEC mH F – ATGAAACGAGTTATTACCCTGTTTG / R – TTATTGATAAACAAAAGTCACGCC 903 Set 3
E. coli EPEC AIDAI F – TGGTGGGAAAACCACTGCTA / R – TAGCCGCCATCACTAACCAG 771 Set 3
E. coli EPEC pAA F – CCATAAAGACAGCTTCAGTGAAAA / R – GTATTACTGGTACCACCACCATCA 162 Set 3
E. coli EAEC aggR F – TTAAAATAAGTCAARAATTGTTTTGGTGTTA / R – ATTATAAAAATTAACAATATCAGAATACATCAGTACAC 715 Set 4
E. coli EIEC ipaH F – CCTTTTCCGCGTTCCTTGA / R – CAGCAGCAACAGCGAAAGAC 104 Set 4
E. coli SepEC cvaC F – TTTCGACACCCCGGTAAAGG / R – TGTCAGTCTGGTTTACGGGC 242 Set 4
TABLE II
Calf No
Age
(days)
Fever Dehydration Feces
Test Result
1 11 39.9 +++
Mucoid yellow
diarrhea
Rotavirus +
Cryptosporidium spp.
2 15 39.7 +++
Mucoid
diarrhea
Rotavirus +
Cryptosporidium spp.
3 12 40.2 +++
Mucoid yellow
diarrhea
Rotavirus +
Cryptosporidium spp.
4 11 41 +++
Mucoid
diarrhea
Rotavirus
5 14 39.8 +++
Mucoid
diarrhea
Rotavirus
6 15 40 +++
Mucoid yellow
diarrhea
Rotavirus +
Cryptosporidium spp.
7 4 39.5 +++
Mucoid yellow
diarrhea
E. coli K99
8 3 39.6 +++
Mucoid yellow
diarrhea
E. coli K99
9 10 39.2 +++
Mucoid
diarrhea
Rotavirus
10 9 39.6 +++
Mucoid
diarrhea
Rotavirus
strains showed S colony characteristics. It was determined that the
E. coli strains isolated from feces (numbered 4, 5, 7, and 8) showed
white color growth on sorbitol MC agar, which is characteristic of
E. coli O157 (EHEC) (TABLE III). This feature was not detected in any
E. coli strains isolated from blood. In another study, researchers
found that 7% of calves with bacteremia symptoms showed growth
from blood cultures, and 20% of the positive blood cultures were
identied as E. coli [35]. However, in another study on this subject,
researchers emphasized that 80% of septicemia was caused by E. coli
[37]. These researchers reported that in addition to E. coli, Salmonella
spp., Enterobacter aerogenes, Campylobacter fetus, Klebsiella spp.,
Streptococcus dysgalactiae, and Trueperella pyogenes were isolated
from blood cultures of septicemic calves [35, 37].
E. coli strains are serologically classied according to the antigenic
differences of the 173 O (somatic) and 56 H (agellar) antigens, based
on the typing scheme according to the rule laid down by Kauffmann
in 1947 [38]. E. coli isolated from blood 6/10 (4, 5, 6, 8 and 9, 10) carry
OK3 (O86, O114, O121, O126, O142) group somatic antigens. In calves
1 and 3, fecal and blood – isolated E. coli carry the O33 (2/10) somatic
antigen. Animal number 3 also carries the E. coli K99 mbriae antigen
isolated from feces. E. coli isolated from the blood of the same calf
______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34307
5 of 11
does not have this feature. E. coli isolated from the blood of calf
number 2 carries the somatic antigen O78 (1/10). E. coli isolated from
the blood of calf number 7 has an O8 (1/10) antigen feature. It was
determined that E. coli strains isolated from the feces of calves 7 and
8 calves contained K99 mbriae antigen. Thirty percent of the strains
were negative for somatic antigen and capsular antigen. The strains
isolated from the feces and blood of calf number 1 showed different
somatic antigen characteristics (TABLE IV). In another study, it was
determined that ExPEC strains isolated from Pigs carried somatic
antigens O161, O8, O11, O138, O101 and O26 [39]. Although the O somatic
antigen is generally used for typing ETEC strains [40], it has been
tried to determine which somatic antigen the SepEC strains have.
Isolated E. coli strains were tested with penicillin, B–lactam
combination, 3rd generation cephalosporin, carbapenem,
aminoglycoside, macrolide, tetracycline, quinolones, folate
antagonists according to CLSI 2022 data. Of the 20 E. coli strains
(ten from blood and ten from feces) isolated, three fecal and eight
blood cultures isolated were MDR, and seven fecal and ve blood
cultures isolated were XDR. None of the E. coli isolates are PDR.
E. coli strains isolated from both the feces and blood of animals 1,
7, and 8 were found to be XDR. E. coli strains of animals 2 and 6 were
found as MDR. Differences were observed in antibiotic susceptibility
of E. coli isolated from the blood and feces of animals 3, 4, 5, 9, and
10. While E. coli (SepEC) isolated from the blood of animals 3 and 4
were XDR, E. coli strains isolated from the same animals' feces were
detected as MDR. In animals 5, 9, and 10, E. coli isolated from their
feces was detected as XDR, while E. coli isolated from the blood of
the same animals was detected as MDR.
TABLE III
Biochemical properties and colony structure of E. coli isolated
Strains
Colony
Morphology
Sorbitol
MC
MC EMB
Blood
Agar
Hemolysis
ATCC 31616 S – + + + –
ATCC 43895 S + + + + –
ATCC 25922 S – + + + –
1D* S – + + + –
1K* S – + + + –
2D S – + + + –
2K S – + + + –
3D S – + + + –
3K S – + + + –
4D S + + + + –
4K S – + + + –
5D S + + + + –
5K S – + + + –
6D S – + + + –
6K S – + + + –
7D S + + + + –
7K S – + + + –
8D S + + + + –
8K S – + + + –
9D S – + + + –
9K S – + + + –
10D S – + + + –
10K S – + + + –
*D:
E. coli isolated from fecal origin, *K: E. coli isolated from blood origin
TABLE IV
Evaluation of isolated E. coli
OK1 OK2 OK3 K99 O1 O2 O8 O9 O33 O38 O78 O101
ATCC 31616 – – – + – – – + – – – –
ATCC 43895 + – – – – – – – – – – –
ATCC 25922 – – – – – – – – – – – –
1D* – – – – – – – – + – – –
1K* – – – – – – – – + – – –
2D – – – – – – – – – – – –
2K – – – – – – – – – – + –
3D – – – + – – – – + – – –
3K – – – – – – – – + – – –
4D – – – – – – – – – – – –
4K – – + – – – – – – – – –
5D – – – – – – – – – – – –
5K – – + – – – – – – – – –
6D – – – – – – – – – – – –
6K – – + – – – – – – – – –
7D – – – + – – – – – – – –
7K – – – – – – + – – – – –
8D – – – + – – – – – – – –
8K – – + – – – – – – – – –
9D – – – – – – – – – – – –
9K – – + – – – – – – – – –
10D – – – – – – – – – – – –
10K – – + – – – – – – – – –
*D:
E. coli isolated from fecal origin, *K: E. coli isolated from blood origin
T:D: E. coli K: E. coli DNA isolated from blood
Characteristics of bacteria isolated in calves with septicemia-diarrhea symptom / Uslu et al. ______________________________________
6 of 11
All E. coli strains isolated in the study were found to have resistance
properties to antimicrobial agents (TABLE V). All isolates were
susceptible only to imipenem, which is the carbapenem group. It
was determined that the only antimicrobial agent that could be
recommended for treatment was 3rd generation cephalosporins.
In general, the SepEC group is more resistant than the InPEC group.
The bacteria sharing the same ora acquire such different resistance
mechanisms, which is explained by the fact that the SepEC group
has gene plasticity. Most of the isolates obtained are XDR, and the
remainder are MDR. In another study, it was reported that MDR and
XDR strains were isolated from E. coli isolated from calf diarrhea [41].
It is clear that antibiotics are needed to ght bacteria; therefore,
antibiotic resistance is a critical issue in line with the concept of
one health [42]. The use of most antibiotics for the treatment of
E.coli diarrhea in calves shows the development of resistance due
to malpractices since the effective therapeutic concentration is not
reached in the intestine. In addition, there are calves with a maximum
of 15 d of age in the study group.
DNA samples isolated from bacteria and stool were evaluated by Set
1 multiplex PCR for the gene encoding F4 (715 bp), F5 (222 bp), F6 (589
bp), F18 (441 bp), F41 (941 bp) mbriae antigens. Only the positive control
with E. coli O9 K99 somatic characteristics, ATCC 31616, was genetically
positive for the F5 (K99) mbriae antigen 222 bp gene region (FIG. 2).
All E. coli isolated from fecal DNA, blood, and stool – causing diarrhea
and septicemia were similarly negative for ETEC gene characteristics.
TABLE V
Antibiotic resistance results of isolated E. coli strains according to CLSI 2022
1D*
R R S S R S R R R R
1K*
R R R S R S R R R R
2D
R R S I R S R R R R
2K
R R S S R S S S S R
3D
R R S S R S R S R S
3K
R R R R R S R R R R
4D
R R S S R S R S R R
4K
R R S S R S R R R R
5D
R R R R R S R R R S
5K
R R S S R S R R I S
6D
R R R S R S R S R S
6K
R R R R R S R S R S
7D
R R R R R S R R R R
7K
R R R R R S R R R R
8D
R R R R R S R R R S
8K
R R R R R S R R R R
9D
R R S R R S R R R R
9K
R R S S R S R R I R
10D
R R R S R S R R R R
10K
R R S S R S R R R S
P: Penicillin, AMC: Amoxacillin, CFP: Cefoperazone, CRO: Ceftriaxone, TE: Tetracycline, IPM: Imipenem, SXT: Trimethoprim/sulfamethoxazole,
CN: Gentamicin, ENR: Enrooxacin, E: Erythromycin.*D:
E. coli isolated from fecal origin, *K: E. coli isolated from blood origin
E. coli
T:D: E. coli K: E. coli DNA isolated from blood
______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34307
7 of 11
The DNA samples were evaluated by Set 2 multiplex PCR for
enterotoxin – encoding genes such as STa (229 bp), STb (480 bp), LT
(605 bp), Stx2e (351 bp) and EAST1 (111 bp). Only the positive control,
ATCC 43895 E. coli O157, was positive for the 351 bp gene region
encoding the Shiga toxin named Stx2e (FIG. 3). E. coli O157 EHEC
strain exists as a reservoir of cattle, so products of animal origin are
known to cause human food infection. In addition, studies indicate
that EHEC strains can infect calves in less than 36 h, even though they
have asymptomatic reservoirs in adult cattle [43]. Although some
bacteria (fecal samples of calves 4, 5, 7 and 8) show EHEC features
biochemically, it has been determined that they do not have these
features serotypically and genetically. In addition, it was determined
that SepEC colonies isolated from the blood of these animals did not
form white colonies on Sorbitol MC agar. It was thought that they
did not have O157 somatic antigen because they did not acquire a
pathogenic form in which this feature could develop due to being
a reservoir.
Molecularly, for the detection of genes responsible for adhesin
with Set 3 primers, type 1 pilus (mA 443 bp and mH 903 bp), the
adhesin gene AIDA – I (771 bp) related to diffuse adhesion, and pAA
(162 bp) gene regions for A/E lesions were evaluated. Samples with
positive mA and mH gene regions simultaneously were evaluated
as type 1 pilus positive. The mA and mH genes, which encode type 1
pilus antigen, were found to be positive at the same time in all agents
causing septicemia (SepEC). The positive control, E. coli O9 K99 ATCC
31616 (PC1) type 1 pilus was found positive. Non – pathogenic E. coli
ATCC 25922 (PC2) was found positive only for the mA gene. For this
reason, it was evaluated as negative for type 1 pilus antigen (FIG. 4).
ATCC 43895 (PC3) with E. coli O157 was found to be positive in terms
of type 1 pilus and pAA gene region causing A/E lesion (FIG. 4). ExPEC
is known to have a wide variety of virulence factors; Adhesins such as
Type 1 mbriae, P mbriae, S mbriae, invasin such as Ibe ABC, iron
uptake antigens such as aerobactin, intracellular survival antigen such
as outer membrane protein and colonization factors such as cvaC
colicinV and cytotoxins [12, 44]. In a previous study with calves with
septicemia, the virulence genes of SEPEC isolates were examined,
and aerobactin was found in 88%, and the gene responsible for 80%
of mbriae – associated adhesion [37]. In the study, type 1 mbriae
set 3 multiplex PCR, responsible for its adhesion, and cvaC gene,
responsible for SEPEC colonization, were studied in set 4 multiplex
PCR. It was determined that 10/10 of the SepEC strains examined in
this study carried type 1 mbriae, and 9/10 had the cvaC gene.
Set 4 multiplex PCR prepared for the last two pathotypes causing
intestinal infections (InPEC), EAEC (aggR 715 bp gene region), EIEC
(ipaH 104 bp), and for the detection of extra – intestinal infections
ExPEC, SepEC pathotype, colicin V gene region cvaC 242 bp for all
samples tested. All samples and control DNAs were molecularly
negative for EAEC and EIEC. Positive controls and all samples were
positive for the colicin gene region. Only all fecal DNA obtained from
sample number 10 and E. coli DNA isolated from feces and blood
culture were negative for the colicin V (cvaC) gene region (FIG. 5). EAEC
and EIEC strains were not isolated in the study. These pathotypes
have often been associated with human infections.
In other studies, the ratio of EAEC (0.9%) and EIEC (0%), EPEC
(2.9%), and ETEC (1.9%) was isolated from the feces of 113 newborn
calves [45]. It has been reported that the signicant source of EIEC
infection for humans is an infection of fecal – oral origin, with chronic
diarrhea generally seen in underdeveloped Countries. No animal
reservoir has been reported before [46]. The Colicin V gene is among
the virulent factors in the ExPEC bacterial group. Another nding in
T:D: E. coli
K: E. coli
T: Whole
D: E. coli K: E. coli DNA isolated from blood
Characteristics of bacteria isolated in calves with septicemia-diarrhea symptom / Uslu et al. ______________________________________
8 of 11
the study is the presence of plasmid carrying the CvaC gene, which
is the Colicin V gene, in the SepEC strains. Samples were positive for
this gene CvaC, except samples from animal number 10. It is usually
produced at a time of stress encountered by the bacterium. It is
thought to be secreted into the intestinal tract to colonize the surface,
reducing bacterial competition eciently. Colicin has also been
reported to have a toxic effect on eukaryotic cells, and its primary
virulence is formed this way [47]. This gene region has been found in
human SepEC and UPEC cases. It is also known that another ExPEC
group, the Avian Pathogen E. coli (APEC) group bacteria, carries
these virulence features.
In the study, no isolates with EPEC, EHEC, EAEC, and EIEC
morphological biochemical serotype or genetic characteristics could
be detected. Only two ETEC group isolates were obtained from InPEC
E. coli diarrhea. According to the study results, ETEC, rotavirus, and
Cryptosporidium spp. cause infection in the intestinal tract, which
causes diarrhea symptoms, and SepEC causes septicemia.
In the diarrheal feces screened with the bovid – 5ag test, animal
numbers 7 and 8 were positive for E. coli F5 and were found to carry F5
mbriae antigen by serotyping test. However, it was determined that
the same isolates did not genetically carry the F5 gene. In addition,
it was determined that SepEC isolates that cause septicemia in
the blood did not carry the K99 antigen in serotyping. Another
current concept in E. coli infections is the presence of hybrid or
heteropathogenic E. coli strains. Heteropathogenic strains such as
EPEC/ETEC, ExPEC/STEC, and ExPEC/EPEC have been reported
______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34307
9 of 11
to cause more severe infections [48]. It has also been reported that
these strains are prone to genetic change [49]. While there was an
ETEC group carrying K99 in feces in this study, the absence of K99
mbriae in SepEC isolates isolated from blood shows that the ETEC/
ExPEC group may be a heteropathogenic infection derivative. ExPEC
strains carry different combinations of virulence factors such as P
mbriae, S/F1C mbriae subunits, Dr – antigen – binding adhesins,
aerobactin receptor, and group 2 capsule synthesis, and colicin V [50].
The most important condition of ExPEC infections is colonization of
the intestinal wall. In the study, SepEC group microorganisms, due to
the pathogenesis and deterioration in peristaltic movements caused
by infections caused by ETEC, rotavirus, and Cryptosporidium spp. in
the intestinal tract, provide colonization by providing adhesion with
type 1 pilus. It was also determined that SepEC isolates may carry
O8, O33, O78 and OK3 (O86, O114, O121, O126, O142) group somatic
antigens. Notably, 6/10 of SepEC isolates carry the OK3 group somatic
antigen. Except for animal number 8, the common feature of this
group is that the primary infection is caused by rotavirus. It is thought
that the SepEC group isolates present in the ora as a result of the
damage caused by ETEC, rotavirus / Cryptosporidium spp. infection
in enterocytes performs pathogenicity with the aforementioned
somatic antigens (adhesin). This view is also supported by the set 3
multiplex PCR and rapid lateral ow test results. The positive presence
of type 1 mbriae and Colicin V gene regions in DNA isolated from
whole feces indicates that SepEC agents are opportunistic in the
ora. However, bacteria isolated from feces exhibit morphologically
different characteristics from SepEC isolates. This condition may be
related to the change in the antigenic structure of the bacteria as
they cross the intestinal mucosa.
CONCLUSIONS AND IMPLICATIONS
While all calf diarrhea studies or eld treatments focus on InPEC
diarrhea, such as ETEC and EPEC, the opportunistic pathogen SepEC
bacteria are overlooked. As stated in the working hypothesis, it was
determined that rotavirus and Cryptosporidium spp. was primary
agent for diarrhea. While ETEC group E. coli caused less damage to
the intestinal mucosa, and it was observed that the pathogenesis was
made by the opportunistic pathogen SepEC group. This result shows
that the generalization about the same agent in calf diarrhea causing
enteric infection with septicemia should be abandoned. Rapid lateral
ow tests for calf diarrhea should be used for early diagnosis and
successful treatment in farm eld conditions. Interestingly, although
the SepEC originates from the intestinal ora, it has been observed
to have different morphological, antibiotic resistance, serotype, and
genetic features from InPEC. E. coli vaccine studies, generally done
in cattle, focus on ETEC and EPEC groups.
In addition to these factors, it has been determined that the
antigenic structures responsible for type 1 pilus adhesion of the
SepEC group, which causes severe destruction when it infects the
organism, should be examined more closely and that these antigens
should be included in preventive vaccine studies. In the study, the
antibiotic resistance of bacteria isolated from calves at a maximum
of 15 d of age. This result shows that medical need to take severe
precautions against antibiotic resistance.
ACKNOWLEDGMENTS
This study was supported by Selcuk University, Coordinatorship
of Scientic Research Projects (No. 22401061).
Ethics approval
The study protocol was approved by Ethical Committee of Faculty of
Veterinary Medicine, Selcuk University, Türkiye (Approval no:2022/24).
Conict of interest
The authors declare that they have no conict of interest.
BIBLIOGRAPHIC REFERENCES
[1] Lorenz I, Fagan J, More SJ. Calf health from birth to weaning.
II. Management of diarrhoea in pre – weaned calves. Ir. Vet. J.
[Internet]. 2011; 64(9):1 – 6. doi: https://doi.org/d3vqkr
[2] Hur TY, Jung YH, Choe CY, Cho YI, Kang SJ, Lee HJ, Ki KS, Baek
KS, Suh GH. The dairy calf mortality: the causes of calf death
during ten years at a large dairy farm in Korea. Korean J. Vet.
Res. [Internet]. 2013; 53(2):103 – 8. doi: https://doi.org/gmxmmn
[3] Constable PD. Antimicrobial use in the treatment of calf diarrhea.
J. Vet. Intern. Med. [Internet]. 2004; 18(1):8 – 17. doi: https://doi.
org/fwt57r
[4] Pardon B, Alliët J, Boone R, Roelandt S, Valgaeren B, Deprez P.
Prediction of respiratory disease and diarrhea in veal calves
based on immunoglobulin levels and the serostatus for
respiratory pathogens measured at arrival. Prev. Vet. Med.
[Internet]. 2015; 120(2):169 – 76. doi: https://doi.org/f7gbcw
[5] Fecteau G, Van Metre DC, Pare J, Smith BP, Higgins R, Holmberg
CA, Jang S, Guterbock W. Bacteriological culture of blood from
critically ill neonatal calves. Can. Vet. J. 1997; 38(2):95 – 100. Cited
in PUBMED; PMID 9028592
[6] Bartels CJ, Holzhauer M, Jorritsma R, Swart WA, Lam TJ.
Prevalence, prediction and risk factors of enteropathogens
in normal and non – normal faeces of young Dutch dairy calves.
Prev. Vet. Med. [Internet]. 2010; 93(2 – 3):162 – 169. doi: https://
doi.org/ch7mpc
[7] Cho YI, Yoon KJ. An overview of calf diarrhea – infectious
etiology, diagnosis, and intervention. J. Vet. Sci. [Internet].
2014; 15(1):1 – 17. doi: https://doi.org/f5wkwh
[8] Kaper JB, Nataro JP, Mobley HL. Pathogenic Escherichia coli.
Nat. Rev. Microbiol. [Internet]. 2004;2(2):123 – 140. doi: https://
doi.org/bb2pfn
[9] Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin.
Microbiol. Rev. [Internet]. 1998;11(1):142 – 201. doi: https://doi.
org/gm8zp8
[10] Foster D, Smith GW. Pathophysiology of diarrhea in calves. Vet.
Clin. N. Am. – Food Anim. Pract. [Internet]. 2009; 25(1):13 – 36.
doi: https://doi.org/cpxrvm
[11] Francis D, Allen S, White R. Inuence of bovine intestinal uid
on the expression of K99 pili by Escherichia coli. Am. J. Vet. Res.
1989; 50(6):822 – 826. Cited in PUBMED; PMID 2569853
[12] Sarowska J, Futoma – Koloch B, Jama – Kmiecik A, Frej – Madrzak
M, Ksiazczyk M, Bugla – Ploskonska G, Choroszy – Krol I. Virulence
factors, prevalence and potential transmission of extraintestinal
pathogenic Escherichia coli isolated from different sources:
recent reports. Gut Pathog. [Internet]. 2019; 11(10):1 – 16. doi:
https://doi.org/gg5pns
Characteristics of bacteria isolated in calves with septicemia-diarrhea symptom / Uslu et al. ______________________________________
10 of 11
[13] Köhler CD, Dobrindt U. What denes extraintestinal pathogenic
Escherichia coli? Int. J. Med. Microbiol. [Internet]. 2011;
301(8):642 – 647. doi: https://doi.org/dwpgdb
[14] Bertin Y, Martin C, Girardeau JP, Pohl P, Contrepois M. Association
of genes encoding P mbriae, CS31A antigen and EAST 1 toxin
among CNF1 – producing Escherichia coli strains from cattle with
septicemia and diarrhea. FEMS Microbiol. Lett. [Internet]. 1998;
162(2):235 – 239. doi: https://doi.org/d8dgvm
[15] Watts RE, Totsika M, Challinor VL, Mabbett AN, Ulett GC, De
Voss JJ, Schembri MA. Contribution of siderophore systems
to growth and urinary tract colonization of asymptomatic
bacteriuria Escherichia coli. Infect. Immun. [Internet]. 2012;
80(1):333 – 344. doi: https://doi.org/cqfdf2
[16] Fecteau G, Smith BP, George LW. Septicemia and meningitis in
the newborn calf. Vet. Clin. N. Am. – Food Anim. Pract. [Internet].
2009; 25(1):195 – 208. doi: https://doi.org/c3hn3h
[17] Minassian AM, Newnham R, Kalimeris E, Bejon P, Atkins BL, Bowler
IC. Use of an automated blood culture system (BD BACTEC™) for
diagnosis of prosthetic joint infections: easy and fast. BMC Infect.
Dis. [Internet]. 2014; 14:1 – 7. doi: https://doi.org/gb3z5j
[18] Moxley R. Enterobacteriaceae: Escherichia. In: McVey DS,
Kennedy M, Chengappa MM, editors. Veterinary Microbiology.
3rd ed. Ames, Iowa, USA: John Wiley & Sons; 2013; p 62 – 74.
[19] Hadimli HH, Pinarkara Y, Sakmanoğlu A, Sayin Z, Erganiş O, Uslu
A, Al – Shattrawi HJ. Serotypes of Salmonella isolated from feces
of cattle, buffalo, and camel and sensitivities to antibiotics in
Turkey. Turkish J. Vet. Anim. Sci. [Internet]. 2017; 41(2):193 – 198.
doi: https://doi.org/k5hk
[20] Lassen J. Rapid identication of Gram‐negative rods using
a three‐tube method combined with a dichotomic key. Acta
Pathol. Microbiol. Scand. B. [Internet]. 1975; 83(6):525 – 533.
doi: https://doi.org/cc2r
[21] Scheutz F, Cheasty T, Woodward D, Smith HR. Designation of
O174 and O175 to temporary O groups OX3 and OX7, and six new
E. coli O groups that include Verocytotoxin‐producing E. coli
(VTEC): O176, O177, O178, O179, O180 and O181. APMIS. 2004;
112(9):569 – 84. doi: https://doi.org/cgtmh2
[22] McFarland J. The nephelometer: an instrument for estimating
the number of bacteria in suspensions used for calculating the
opsonic index and for vaccines. J. Am. Med. Assoc. [Internet].
1907; 49(14):1176 – 1178. doi: https://doi.org/csdkhx
[23] CLSI. Performance standards for antimicrobial susceptibility
testing. 32nd. ed. CLSI supplement M100. Clinical and Laboratory
Standard Institute; 2022; 402 p.
[24] Lee SI, Kang SG, Kang ML, Yoo HS. Development of multiplex
polymerase chain reaction assays for detecting enterotoxigenic
Escherichia coli and their application to eld isolates from
piglets with diarrhea. J. Vet. Diagn. Investig. [Internet]. 2008;
20(4):492 – 496. doi: https://doi.org/bhw3p9
[25] Oh KH, Kim SB, Park MS, Cho SH. Development of a one – step
PCR assay with nine primer pairs for the detection of five
diarrheagenic Escherichia coli types. J. Microbiol. Biotechnol.
[Internet]. 2014; 24(6):862 – 868. doi: https://doi.org/k5hm
[26] Vandekerchove D, Vandemaele F, Adriaensen C, Zaleska M,
Hernalsteens JP, De Baets L, Pasmans F. Virulence – associated
traits in avian Escherichia coli: comparison between isolates
from colibacillosis – affected and clinically healthy layer ocks.
Vet. Microbiol. [Internet]. 2005; 108(1 – 2):75 – 87. doi: https://
doi.org/ft63sz
[27] Aydin F, Umur Ş, Gökçe G, Genç O, Güler MA. [The Isolation and
Identication of Bacteria and Parasites from Diarrhoeic Calves in
Kars District]. Kafkas Univ. Vet. Fak. Derg. [Internet]. 2001 [cite 20
Jun 2023]; 7(1):7 – 14. Turkish. Available in: https://bit.ly/3unUPSw.
[28] Brunauer M, Roch FF, Conrady B. Prevalence of worldwide neonatal
calf diarrhoea caused by bovine rotavirus in combination with
Bovine Coronavirus, Escherichia coli K99 and Cryptosporidium
spp.: A Meta – Analysis. Anim. [Internet]. 2021; 11(4):1014. doi:
https://doi.org/gnqsw3
[29] Işik – Uslu N, Derinbay – Ekici O, Avci O. ELISA – based Point
Prevalence of enteropathogens in diarrheic calves in Central
Anatolia Region of Turkey. Rev. Cient. Fac. Cien. Vet. Univ. Zulia.
[Internet]. 2023; 33(2):1 – 6. doi: https://doi.org/k5hn
[30] Içen H, Arserim NB, Işik N, Özkan C, Kaya A. Prevalence of Four
Enteropathogens with Immunochromatographic Rapid Test in
the Feces of Diarrheic Calves in East and Southeast of Turkey.
Pak. Vet. J. [Internet]. 2013 [cited 20 Jun 2023]; 33(4):496 – 499.
Available in: https://bit.ly/3R8utvm.
[31] Cengiz S, Adiguzel MC. Determination of virulence factors and
antimicrobial resistance of E. coli isolated from calf diarrhea,
part of eastern Turkey. Ankara Univ. Vet. Fak. Derg. [Internet].
2020; 67(4):365 – 371. doi: https://doi.org/kdr6
[32] Küliğ C, Coşkun A. [Prevalence of E. coli, Cryptosporidium,
Clostridium perfringens, Rotavirus and Coronavirus in Neonatal
Calves with Diarrhea in Sivas]. Turk. Vet. J. [Internet] 2019
[cited 21 Jun 2023]; 1(2):69 – 73. Turkish. Available in: https://
bit.ly/3GbBBSt.
[33] Güler L, Gündüz K, Ok Ü . Virulence factors and antimicrobial
susceptibility of Escherichia coli isolated from calves in Turkey.
Zoon. Publ. Health. [Internet]. 2008; 55(5):249 – 257. doi: https://
doi.org/dwhbkb
[34] Altuğ N, Yüksek N, Özkan C, Keleş İ, Başbuğan Y, Ağaoğlu ZT,
Kaya A, Akgül Y. [Rapid Etiological Diagnosis of Neonatal Calf
Diarrhoea with Immunochromatographic Test Kits]. YYU Vet.
Fak. Derg. [Internet] 2013 [cited 15 Jun 2023]; 24(3):123 – 128.
Turkish. Available in: https://bit.ly/3MQRbqx.
[35] Garcia J, Pempek J, Hengy M, Hinds A, Diaz – Campos D, Habing
G. Prevalence and predictors of bacteremia in dairy calves with
diarrhea. J. Dairy Sci. [Internet]. 2022; 105(1):807 – 817. doi:
https://doi.org/k5hp
[36] Lofstedt J, Dohoo IR, Duizer G. Model to predict septicemia in
diarrheic calves. J. Vet. Intern. Med. [Internet]. 1999; 13(2):81 – 88.
doi: https://doi.org/fnqg4w
[37] Uriarte ELL, Pasayo RAG, Masso M, Paez LC, Moncla MD, Donis
N, Malena R, Méndez A, Morrell E, Giannitti F, Armendano
JI, Faverin C, Centrón D, Parreño V, Odeón AC, Quiroga MP,
Moreira AR. Molecular characterization of multidrug – resistant
______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34307
11 of 11
Escherichiacoli of the phylogroups A and C in dairy calves with
meningitis and septicemia. Microb. Pathog. [Internet]. 2022;
163:105378. doi: https://doi.org/k5hq
[38] Kauffmann F. The serology of the coli group. J. Immunol.
[Internet]. 1947; 57(1):71 – 100. doi: https://doi.org/k5hr
[39] Tan C, Tang X, Zhang X, Ding Y, Zhao Z, Wu B, Cai X, Liu Z, He Q, Chen
H. Serotypes and virulence genes of extraintestinal pathogenic
Escherichia coli isolates from diseased pigs in China. Vet. J.
[Internet]. 2012; 192(3):483 – 488. doi: https://doi.org/bwns5h
[40] Debroy C, Roberts E, Fratamico PM. Detection of O antigens
in Escherichia coli. Anim. Health Res. Rev. [Internet]. 2011;
12(2):169 – 185. doi: https://doi.org/cw8q6w
[41] Feuerstein A, Scuda N, Klose C, Hoffmann A, Melchner A, Boll
K, Rettinger A, Fell S, Straubinger RK, Riehm JM. Antimicrobial
resistance, serologic and molecular characterization of E. coli
isolated from calves with severe or fatal enteritis in Bavaria,
Germany. Antibiot. [Internet]. 2021; 11(1):23. doi: https://doi.
org/k5hs
[42] World Health Organization. Antimicrobial resistance: global
report on surveillance. [Internet]. Geneva: WHO. 2014 [cited
18 Apr 2023]. 232 p. Available in: https://bit.ly/3R0VrWV.
[43] Lange ME, Uwiera RR, Inglis GD. Enteric Escherichia coli O157: H7
in Cattle, and the Use of Mice as a Model to Elucidate Key Aspects
of the Host – Pathogen – Microbiota Interaction: A Review. Front.
Vet. Sci. [Internet]. 2022; 9:937866. doi: https://doi.org/gsndw5
[44] Sora VM, Meroni G, Martino PA, Soggiu A, Bonizzi L, Zecconi A .
Extraintestinal pathogenic Escherichia coli: Virulence factors
and antibiotic resistance. Pathog. [Internet]. 2021; 10(11):1355.
doi: https://doi.org/k5jh
[45] Khawaskar DP, Sinha DK, Lalrinzuala MV, Athira V, Kumar M,
Chhakchhuak L, Thomas P. Pathotyping and antimicrobial
susceptibility testing of Escherichia coli isolates from neonatal
calves. Vet. Res. Commun. [Internet]. 2022; 46(2):353 – 362. doi:
https://doi.org/k5jj
[46] Pasqua M, Michelacci V, Di Martino ML, Tozzoli R, Grossi M,
Colonna B, Morabito S, Prosseda G. The intriguing evolutionary
journey of enteroinvasive E. coli (EIEC) toward pathogenicity.
Front. Microbiol. [Internet]. 2017; 8:2390. doi: https://doi.org/
gcrbhw
[47] Šmajs D, Micenková L, Šmarda J, Vrba M, Ševčíková A, Vališová
Z, Woznicová V. Bacteriocin synthesis in uropathogenic and
commensal Escherichia coli: colicin E1 is a potential virulence
factor. BMC Microbiol. [Internet]. 2010; 10(1):1 – 10. doi: https://
doi.org/c3gtfm
[48] Santos ACDM, Santos FF, Silva RM, Gomes TAT. Diversity of
hybrid – and hetero – pathogenic Escherichia coli and their
potential implication in more severe diseases. Front. Cell. Infect.
Microbiol. [Internet]. 2020; 10:339. doi: https://doi.org/k5jk
[49] Pokharel P, Dhakal S, Dozois CM. The diversity of Escherichia
coli pathotypes and vaccination strategies against this versatile
bacterial pathogen. Microorgan. [Internet]. 2023; 11(2):344. doi:
https://doi.org/k5jm
[50] Johnson JR, Murray AC, Gajewski A, Sullivan M, Snippes P,
Kuskowski MA, Smith KE. Isolation and molecular characterization
of nalidixic acid – resistant extraintestinal pathogenic Escherichia
coli from retail chicken products. Antimicrob. Agents Chemother.
[Internet]. 2003; 47(7):2161 – 2168. doi: https://doi.org/fjxvjv
