Overall this paper is well written, well presented and referenced. The majority of
the editorial changes included below are grammatical and intended to help the
authors secure publication. However, I have two major comments on the content,
which could be rectified via obtaining more field data and adding it to this paper
and modifying the statistaical method.
1.
There is a need for additional data to make the conclusions more robust.

It is not realistic to base a conclusion of antibiotic resistance on 2 E.coli
isolates. There are too few of all other isolates too.

Most isolates were A.pyogenes (18) even so an MIC90 would need an
absolute minimum of 20 isolates. Any statement of antibacterial
resistance even from the simple disk diffusion method (rather than a
titration method) needs to be expressed as percent of resistant isolates
from a meaningful total number of tested isolates.
2. Need to clarify how reproductive tract structural abnormalities are graded
and use a statistical method to correlate these with bacterial isolation. I
suggest the easiest way would be to use a rank system (e.g. 1-4) then use
either Pearson’s or Spearman’s Rank testing for strength of correlation.
If the authors could obtain a substantial additional amount of isolates, use a
statistical method to assess strength of correlation to support their conclusions and
make the suggested grammatical changes this paper could then become suitable
for publication.
Bacterial study of clinical postpartum endometritis in
Holstein dairy cows
Morteza Yavari1, Masoud Haghkhah2, Mohammad Rahim Ahmadi1
Department of Clinical Sciences1 and Pathobiology2 School of Veterinary Medicine,
Shiraz University, P.O.Box:71345-1731, Shiraz, Iran
a
Corresponding Author:
Mohammad Rahim Ahmadi
Professor of Theriogenology
Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University,
PO Box: 71345-1731, Shiraz, Iran.
Mobile: +98-917-700-1074
Fax: +98-711-228-6950
E-mail: rahmadi@shirazu.ac.ir
Abstract
Endometritis is inflammation of the endometrial lining of the uterus and is
associated with delayed uterine involution and poor reproductive performance. The aim
of this study was to present the results of bacteriological culture from uterine swabs of
dairy cows affected with postpartum endometritis and to evaluate the antimicrobial
susceptibility of the isolated bacteria. In total, eighty nine Holstein cows affected with
postpartum endometritis were selected and sampled between 21-35 day postpartum.
Swabs (n=89) were collected from the uterine lumen of dairy cattle. Bacteria were
identified following aerobic and anaerobic culture and the disk diffusion method was
used to determine susceptibility of the major pathogenic isolated bacteria. The results
revealed that the most common isolates from cases of endometritis in study cows were
Arcanobacterium pyogenes, E. coli, and non-differentiated streptococci, staphylococci
and bacilli (type?). The antimicrobial susceptibility tests showed that E. coli were
sensitive to enrofloxacin and ceftiofur (only 2 isolated means nothing), but resistant to
tetracycline and oxytetracycline. For A. pyogenes, 72, 66, 72 and 72 percent of isolates
were resistant to oxytetracycline, tetracycline, enrofloxacin and ceftiofur respectively.
All isolates showed resistance to penicillin. In conclusion, oxytetracycline which is the
most traditional antimicrobial therapy for postpartum endometritis in cows appears not
to be efficacious. There is widespread resistance to enrofloxacin and third generation
cephalosporins too. Therefore, dairy farms need to evaluate alternatives to treating and
preventing post-partum endometritis including non antibiotic options.
Keywords: Endometritis; Bacteria; Antimicrobial susceptibility; Uterus; Dairy cow
Abbreviations:
EPC=Epithelial cells
LVEP=large vacuolated epithelial cells
MAC=macrophage
EN=endometritis
Neut=neutrophils
Lym=lymphocytes
LH= Lutenizing hormone
C=Cervix
Introduction
During parturition, the physical barriers of the cervix, vagina and vulva are
compromised providing the opportunity for bacteria to ascend the genital tract. Bacteria
can be isolated from the uterus of over 90% of cows early postpartum (Griffin et al.,
1974; Paisley et al., 1986). Most healthy cows are able to clear the uterus of bacteria
within the first 2 to 3 weeks after calving (Bondurant, 1999). However, cows that
cannot eliminate the infection may subsequently develop endometritis (Dhaliwal et al.,
2001).
Endometritis is a common reproductive disorder in female domestic animals with
consequences ranging from no effect on reproductive performance to permanent
sterility. It affects the general health of animals and adversely affects their reproductive
performance (Amiridis et al., 2003). Subclinical endometritis, based on uterine
cytological examination, is also prevalent in dairy cows and has a profound negative
impact on reproductive performance (Hammon et al., 2006). The presence of bacteria in
the uterus causes inflammation, histological lesions of the endometrium and delays
uterine involution (Bonnett et al., 1991, Sheldon et al., 2003). In addition, uterine
bacterial infection or bacterial products suppress pituitary LH secretion, and perturb
postpartum ovarian follicle growth and function, which disrupts ovulation in cattle
(Sheldon et al., 2002b, Opsomer et al., 2000, Peter and Bosu., 1988, Peter et al., 1989).
Thus, endometritis is associated with lower conception rates, increased intervals from
calving to first service or conception, and more culls for failure to conceive (Borsberry
and Dobson, 1989, Huszenicza et al., 1999, LeBlanc et al., 2002).
A variety of antimicrobial agents, administered by intrauterine infusion or
parenteral injection, are used to treat uterine infections (Cohen et al., 1995). There is
little literature available concerning bacterial causes of postpartum endometritis and
their susceptibility to suitable candidate antibiotics in Iranian dairy farms. The aim of
this study was: (a) to identify isolates by bacteriological culture from uterine swabs of
Holstein dairy cows with clinical endometritis between 21 and 35 days postpartum and
(b) to evaluate the antimicrobial susceptibility of the most common isolates.
MATERIALS AND METHODS
Animals
The study was carried out in 13 large commercial dairy herds of Iran (please give
range of cow numbers). Four hundred two postpartum dairy cows were examined once
between 21 and 35 days postpartum. In total 89 cows affected with clinical endometritis
were selected. Cows in all herds were calved in calving boxes hygienically and kept in
individual boxes for at least 10 days after parturition. Corn silage, alfalfa hay, and
concentrates as a total mixed ration were used. None of the cows received any
intrauterine or reproductive hormonal therapy for at least 10 days before sampling for
this study.
Clinical examination
Cows were first examined for the presence of recent discharge on the vulva,
perineum, or tail. If discharge was not visible externally, cows were examined
vaginally. The cow’s vulva was thoroughly cleaned with a dry paper towel and a clean,
lubricated, gloved hand was inserted into the vagina. In each cow, the lateral, dorsal and
ventral walls of the vagina were palpated, and the mucus contents of the vagina
withdrawn manually for examination, as described by Sheldon et al. (2002a). The
vaginal mucus was assessed for color and presence of pus. The nature of the discharge
was classified as clear mucus, clear mucus with flecks of pus, mucopurulent
(approximately 50% pus and 50% mucus), purulent (>50% pus) but not foul-smelling,
purulent or red-brown and foul smelling using the methodology described by LeBlanc
et al. (2002).
Following vaginal examination, transrectal palpation of the reproductive tract was
performed and cervical diameter, location of the uterus, symmetry of the uterine horns,
diameter of the (larger) uterine horn, texture of uterine wall, palpable uterine lumen,
dominant palpable ovarian structure including corpus luteum (CL), follicle, cyst (>2.5
cm in diameter), or no palpable structures was recorded (LeBlanc et al., 2002). (do you
mean palpable abnormalities? – no structure means absence of tract palpated)
Ultrasonographic assessment of uterus and ovaries using a 5MHz rectal linear
probe (AMI Company, Canada) was also performed. Diameter of the uterus,
echotexture and thickness of the uterine wall and intraluminal fluid accumulation were
evaluated in the cows. Ovarian structures (follicle, CL and cyst) were scanned and
measured by calipers (Mateus et al., 2002).
Uterine swab collection and bacteriological culture
For each animal, a transcervical guarded swab was collected from the uterine body
(Noakes et al., 1989). The swab comprised a long copper wire bearing a cotton wool tip
sheathed in a metal guard tube (8 mm external diameter; 58 cm long) and was wrapped
and sterilized by autoclaving at 121°C for 15 min. The guard tube was covered by a
sterile plastic sheath to prevent contamination of the swab during the cervix insertion.
After restraining the animal and securing its tail, the perineal region was washed
and cleaned. The cervix was grasped per-rectum and the sterilized catheter was passed
through the cervix into the uterine body. Then, the inner rod of the catheter was pushed
forward to expose the swab to the endometrium and was rotated against the uterine wall
and then withdrawn within the catheter. To avoid contamination, the catheter was then
cleaned with alcohol. Swabs were cultured immediately on sheep blood agar and
MacConkey agar (MERCK), and incubated at 37°C for 48 h. The same culture on sheep
blood agar (MERCK) was incubated anaerobically for up to 7 days. Standard
biochemical tests were used for the isolation and identification of the isolates as
described by (Quinn et al. (1994).
Blood sampling and progesterone assays
Blood samples were collected from the coccygeal vein or artery into evacuated
tubes and transported on ice to the laboratory. Serum was separated by centrifugation at
2500 rpm for 10 min and stored frozen at -20°C until required. Plasma progesterone
concentration was measured by radioimmunoassay (Spectria® Progesterone RIA,
Espoo, Finland) with a sensitivity of 0.1 ng/ml and intra- and inter-assay coefficients of
variation of 10.2% and 6.5 % respectively.
Antimicrobial susceptibility tests
According to the categorization of bacteria isolated by culture of uterine swabs,
based on their potential pathogenicity within the uterus (Sheldon, 2004),
Arcanobacterium pyogenes and E. coli were the only major pathogens identified and
used for antibiotic susceptibility tests. Disk diffusion method was performed to
determine susceptibility of the major isolated pathogenic bacteria based on the NCCLS
1996 protocol (Please cite the reference as NCCLS 1996 etc and add to list at end of
paper). The bacterial suspension turbidity adjusted to McFarland standard number 0.5 in
Mueller-Hinton broth (MERCK) and inoculated on Mueller-Hinton agar (MERCK)
with a sterile cotton swab.
Commercial antibiotic disks containing single concentrations of each antibiotic
were then placed onto the inoculated plate surface. Inhibition zone of growth around
each disk after overnight incubation at 37°C, were measured. The zone diameter was
interpreted using a zone size interpretation chart (Lorian, 1996). The antibiotics and
their concentration per disc were as follows: tetracycline 30µg, oxytetracycline 30µg,
penicilin 10 international units (iu), enrofloxacin 5µg and ceftiofur 30µg (Quinn et al.,
1994).
Statistical analysis
Data were analyzed by using SAS software, version 6.12. Association of ovarian
structure (please detail how), progesterone serum level and discharge status (is this
ranked by severity) with bacterial culture results in postpartum endometric cows were
determined by using Chi-square and Fisher exact tests (SAS, 1991).
Results
In total, 89 Holstein cows were selected and sampled at 21-35 day postpartum.
Thirty five (39.3%) swabs were found bacteriologically positive and the remaining 54
(60.7%) showed no bacterial growth. Aerobic and facultative anaerobic bacteria which
were isolated are listed in Table 1. A total of 61 isolates were identified from the
positive swabs. The most frequently isolated facultative anaerobe was A. pyogenes 18
(29.51%) followed by Bacillus spp. 13 (21.31%), Streptococci 8 (13.11%),
Staphylococci (9.83) and Lactobacillus 8 (13.11%). Twenty three (66%) of positive
swabs yielded pure bacterial growth, of which A. pyogenes was the most frequently
isolate (Table 1). Character of uterine discharge and bacterial isolation variables were
dependent and mucus characters had significant different bacterial isolation (P<0.05).
Character of uterine discharge and bacterial isolation variables were dependent and
mucus characters had significant (P<0.05) different bacterial isolation (Table 2).
Ovarian structure and P4 level of serum were not correlated with bacterial isolation
(P>0.05) (Table 3). (Should use Spearmans or Pearson’s rank correlation coefficients)
The result of antimicrobial susceptibility tests showed that both of E. coli
isolates (not enough to be conclusive) were sensitive to enrofloxacin and ceftiofur, but
they were resistant to tetracycline and oxytetracycline (Table 4). For A. pyogenes, (state
number of isolates as not many and % is misleadingly conclusive) 72, 66, 72 and 72
percent of isolates were resistant to oxytetracycline, tetracycline, enrofloxacin and
ceftiofur respectively. All bacteria isolate showed resistance to penicillin (Fig.1).
TABLE 1. Bacterial isolates from postpartum endometritis of dairy cows
Bacteria
A. pyogenes
E. coli
Streptococci (total)
S. equinus
S. dysgalactiae
S. spp
Staphylococci (total)
S. epidermidis
S. spp
Corynebacterium spp
Mannhiema haemolytica
Micrococcus spp
Bacilli (total)
B. coagulans
B. firmus
B. pumilus
B. spp.
Lactobacillus spp
Total
Number
18
2
8
1
1
6
6
1
5
2
1
3
13
3
3
3
4
8
61
%
29.51
3.28
13.11
1.64
1.64
9.83
9.83
1.64
8.19
3.28
1.64
4.92
21.31
4.92
4.92
4.92
6.56
13.11
100
Table 2. Vaginal mucus discharge and bacterial isolation in postpartum endometritis
cows.
Character of mucus
Clear
Mucus with
Mucopurulent
Purulent
total
mucus
flecks of pus
Positive
9(10.11)a 5(5.62)abc
10(11.24)b
11(12.36)c 35(39.33)
Negative
34(38.2)a 11(12.36)abc 7(7.87)b
2(2.25)c
54(60.68)
total
43(48.31) 16(17.98)
17(19.10)
13(14.61) 89(100)
Values with different superscripts in each row are those that differ significantly
(p<0.05)
Bacteriology
Table 3. Association of ovarian structure and plasma progesterone concentration with
results of bacterial culture in postpartum endometritis cows
Bacteriology
Positive
Negative
total
Ovarian Structure
Nos. (%)
No
Follicle
Corpus
structures
luteum
4
22
9
1
35
18
5 (5.62)
57 (64.04) 27 (30.34)
P4
Nos. (%)
< 1ng/ml
> 1ng/ml
26
34
60 (67.4)
9
20
29 (32.6)
(what does negative structure mean??)
Table 4. The antimicrobial susceptibility of the major isolated pathogenic bacteria from
endometritis cows using disk diffusion
Bacteria &
Results
Arcanobacterium pyogenes
(number of isolates = 18)
Antibiotics
oxytetracycline
tetracycline
penicillin
ceftiofor
enrofloxacin
E. Coli
(number of isolates = 2!)
Resistance
%
Intermediate
%
Sensitive
%
Resistanc
e%
Intermediat
e%
Sensitive
%
72
66
100
72
72
17
23
0
0
0
11
11
0
28
28
100
0
100
0
0
0
100
0
0
0
0
0
0
100
100
You do not need both Fig 1 and Table 4 as they show the same data use one or other – if
correlation coefficients calculated preference is probably for the table.
ceftiofur
72
28
enrofloxacin
72
28
penicillin
tetracycline
oxytetracycline
100
66
23
72
Resistent
17
Intermediate
11
11
Sensitive
Fig. 1: Antibiotic susceptibility of isolated A. pyogenes from uterine swabs
Discussion
-10-
The uterine lumen is sterile before parturition and if bacterial invasion occurs,
there is usually resorption of the fetus or abortion (Semambo et al., 1991). During or
shortly after parturition, microorganisms from the animal's environment, skin, and feces
contaminate the uterine lumen (Sheldon, 2004; Sheldon and Dobson, 2004; Földi et al.,
2006). The flora of the postpartum uterus has been shown to be quite variable, and the
results of one sample may not give a full picture of the infection status (Griffin et al.,
1974). However, intrauterine bacterial infection, does not necessarily present as a
clinical manifestation of disease; this is dependant on the immune status of the host
(Földi et al., 2006).
The results of this study revealed that the most common isolates from cases of
endometritis in cattle were A. pyogenes, E. coli (no only 2 is not one of the most
common!), streptococci (8) and staphylococci.(6) The species of isolated bacteria were
similar to those reported in the previous studies (Elliot et al., 1968; Sagartz and
Hardenbrook, 1971; Griffin et al., 1974; Hartigan et al., 1974; Studer and Morrow,
1978; Ball et al., 1984; Messier et al., 1984). – this last sentence cannot be used as a
conclusion there is insufficient data. In our study, the most common isolate was A.
pyogenes. Studies confirmed that most of the clinical and reproductive consequences are
attributed to the presence of certain non-specific pathogens: mainly to A. pyogenes,
either alone or in combination with other bacteria such as E. coli and gram negative
obligate anaerobes (Földi et al., 2006). Isolation of A. pyogenes at the late involution
period (28-35 days after calving) is associated with dramatically decreased reconception rate (Huszenicza et al., 1999). In addition; A. pyogenes was the most
frequent single isolate (40%) in our study. Previous studies have shown A. pyogenes to
be recovered in pure culture more frequently with increased days postpartum (Hartigan
et al., 1974).
-11-
Obligate anaerobes such as Fusobacterium necrophorum and Prevotella spp
were not isolated in this study. This result is in agreement with some of other published
reports. Kaczmarowski et al. (2004) reported that anaerobes were of lower significance
in their examinations. In our opinion, it seems that the inconsistency of the bacterial
isolates in different studies may be related to the different bacterial flora of each area
and country. However, Williams et al. (2005) explained that variation between studies
may include the use of clinically ill animals or the selection of animals for
microbiological examination based on likely uterine disease. In addition, the inclusion
of animals <21 days postpartum in other studies may be important as they have a
different bacterial profile to those calved longer.
The effect of character of vaginal mucus on bacterial isolation was considered
independently in the present study. Positive results from bacterial culture was more
commonly associated with mucopurulent or purulent vaginal mucus (give correlation
co-efficient if you wish to make this conclusion). These observations are in agreement
with the previous result of Williams et al. (2005). They reported that the bacterial
growth densities for recognized and potential pathogens were associated with purulent
vaginal mucus, and higher growth density for recognized pathogens was associated with
mucopurulent mucus character. Pus is formed as a result of bacterial infection by a
mixture of viable and dead neutrophil leucocytes, necrotic tissue and tissue fluid, so it is
not surprising that bacterial growth density of pathogenic bacteria is associated with
purulent vaginal mucus (Williams et al., 2005).
It is important to be able to diagnose the presence of uterine infection to
facilitate timely and appropriate treatment and to quantify the severity of disease, which
allows a prognosis to be given for subsequent fertility. Unfortunately, there is no ‘‘gold
standard’’ for diagnosis of uterine disease, making it difficult to measure the sensitivity
and specificity of clinical definitions (Sheldon et al, 2006). Miller et al. (1980)
-12-
concluded that vaginoscopic examination is a more accurate method for detecting
uterine infections than palpation per-rectum. In the study by LeBlanc et al. (2002), the
reproductive performance of cows with purulent discharge on vaginoscopic examination
was significantly lower than cows with no abnormal discharge. However, vaginoscopy
has often failed to identify all cows that are truly at risk of poor reproductive
performance (Kasimanickam et al., 2004).
Progesterone suppresses uterine immune defenses and predisposes the uterus to
nonspecific infections. These occur most commonly in postpartum animals and may
reduce the reproductive performance of livestock (Lewis 2004). However, in the present
study there was no positive association between ovarian structures and progesteroneserum levels (give statistical evidence/ correlation coefficient please) with bacterial
isolation.
According to the categorization of bacteria isolated by culture of uterine swabs,
based on their potential pathogenicity within the uterus (Sheldon, 2004), A. pyogenes
and E. coli were only used for antibiotic susceptibility tests. The results of antimicrobial
susceptibility tests indicated that A. pyogenes
can be resistant to the common
antimicrobial agents (tetracycline, penicillin, enrofloxacin and ceftiofur) used for
intrauterine treatment in the practice field. This is in agreement with previous reports.
Cohen et al. (1995) also found A. pyogenes isolates inthe uterus of cows with retained
fetal membranes or postpartum endometritis that were not susceptible to
oxytetracycline.
Other studies have also isolated A. pyogenes that is less susceptible to
antimicrobial agents commonly used as feed and water additives used in the United
State agriculture (i.e. tetracycline, macrolide and lincosamide) (Trinth et al., 2002).
Sheldon et al. (2004) also reported the relative inefficiency of oxytetracycline against A.
-13-
pyogenes. However, according to their findings, cephalosporins and the fluroquinolone,
enrofloxacin were effective against all the strains of A. pyogenes tested
In conclusion, use of oxytetracycline, the usual antimicrobial therapy for
postpartum endometritis in Iran, appears not to be efficacious. Dairy farms need to
implement alternative procedures for treatment of postpartum endometritis. According
to the suggestion of Hussain and Daniel (1991), dairy farms may benefit from also
evaluating non antibiotic alternatives for the treatment of postpartum endometritis.
References
1- Amiridis GS, Fthenakis GC, Dafopoulos J, Papanikolaou T, Vrogianni VS (2003),
Use of cefquinome for prevention and treatment of bovine endometritis. Journal of
Veterinary Pharmacology Therapy 26, p 387–390
2- Ball L, Olson JD, Mortimer RG (1984), Bacteriology of the postpartum uterus.
Proceeding of Annual Meeting Society of Theriogenology, p 164-169
3- Bondurant RH (1999), Inflammation in the bovine female reproductive tract. Journal
of Animal Science 77 (Suppl), p 101–110
4- Bonnett BN, Martin SW, Gannon VP, Miller RB, Etherington WG (1991),
Endometrial biopsy in Holstein–Friesian dairy cows. III. Bacteriological analysis
and correlations with histological findings. Canadian Journal of Veterinary
Medicine 55, p 168–173
5- Borsberry S, Dobson H (1989), Periparturient diseases and their effect on
reproductive performance in five dairy herds. Veterinary Record 124, p 217–219
-14-
6- Cohen RO, Bernstein M, Ziv G (1995), Isolation and antimicrobial susceptibility of
A. pyogenes recovered from the uterus of dairy cows with retained fetal membranes
and postparturient endometritis. Theriogenology 43, p 1389–1397
7- Dhaliwal GS, Murray RD, Woldehiwet Z (2001), Some aspects of immunology of
the bovine uterus related to treatments for endometritis. Anim Reprod Sci 67:135152
8- Elliot L, McMahon K, Gier HT, Marion GB (1968) Uterus of the cow after
parturition: bacterial content. American Journal of Veterinary Research 29, p 77–81
9- Földi J, Kulcsar M, Pecsi A, Huyghe B, de Sa C, Lohuis JA, Cox P, Huszenicza G
(2006), Bacterial complications of postpartum uterine involution in cattle. Animal
Reproduction Science 96, p 265-281
10- Griffin JFT, Hartigan PJ, Nunn WR (1974), Non-specific uterine infection and
bovine fertility. I. Infection patterns and endometritis during the first seven weeks
post-partum. Theriogenology 1, p 91–106
11- Hammon DS, Evjen IM, Dhiman TR, Goff JP, Walters JL (2006), Neutrophil
function and energy status in Holstein cows with uterine health disorders.
Veterinary Immunology Immunopathology 113, p 21-29
12- Hartigan PJ, Griffin JFI, Nunn WR (1974), Some observations of Corynebacterium
pyogenes infection of the bovine uterus. Theriogenology 1, p 153-167
13- Hussain AM, Daniel RCW (1991), Bovine endometritis: current and future
alternative therapy. Journal of Veterinary Medicine A 38, p 641–651
14- Huszenicza G, Fodor M, Gacs M, Kulcsar M, Dohmen MJW, Vamos M, Porkolab
L, Kegl T, Bartyik J, Lohuis JACM, Janosi Sz, Szita G (1999), Uterine bacteriology,
resumption of cyclic ovarian activity and fertility in postpartum cows kept in largescale dairy herds. Reproduction Domestic Animal 34, p 237–245
-15-
15- Kaczmarowski M, Malinowski E, Markiewicz H (2004), Influence of various
treatment methods on bacteriological findings in cows with puerperal endometritis.
Polish Journal of Veterinary Science 7, p 171-174
16- Kasimanickam R, Duffield TF, Foster RA, Gartley CJ, Leslie KE, Walton JS
(2004), Endometrial cytology and ultrasonography for the detection of subclinical
endometritis in postpartum dairy cows. Theriogenology 62, p 9–23
17- LeBlanc S, Leslie K, Duffield T, Bateman K, Keefe GP, Walton JS, Johnson WH
(2002), Defining and diagnosing postpartum clinical endometritis and its impact on
reproductive performance in dairy cows. Journal of Dairy Science 85, p 2223–2236
18- Lewis GS (2004), Steroidal regulation of uterine immune defenses. Animal
Reproduction Science 82/83, p 281–294
19- Lorian V (1996), Antibiotics in Laboratory Medicine. 4th ed. Williams and Wilkins
20- Mateus L, Lopes da Costa L, Bernardo F, Silva JR (2002), Influence of puerperal
uterine infection on uterine involution and postpartum ovarian activity in dairy
cows. Reproduction Domestic Animal 37, p 31–35
21- Messier S, Higgins R, Couture Y, Morin M (1984), Comparison of swabbing and
biopsy for studying the flora of the bovine uterus. Canadian Veterinary Journal 25,
p 283-288
22- Miller HV, Kimsey PB, Kendrick JW, Darien B, Doering L, Franti C (1980),
Endometritis of dairy cattle: diagnosis, treatment and fertility. Bovine Practitioner 5,
p 13–23
23- Noakes DE, Till D, Smith GR (1989), Bovine uterine flora post partum: a
comparison of swabbing and biopsy. Veterinary Record 124, p 563–564
24- Opsomer G, Grohn YT, Hertl J, Coryn M, Deluyker H, de Kruif A (2000), Risk
factors for post partum ovarian dysfunction in high producing dairy cows in
Belgium: a field study. Theriogenology 53, p 841–857
-16-
25- Paisley LG, Mickelson WD, Anderson PB (1986), Mechanisms and therapy for
retained fetal membranes and uterine infections of cows: a review. Theriogenology
3, p 353-381
26- Peter AT, Bosu WTK (1988), Relationship of uterine infections and folliculogenesis
in dairy cows during early puerperium. Theriogenology 30, p 1045–1051
27- Peter AT, Bosu WTK, DeDecker RJ (1989), Suppression of preovulatory
luteinizing hormone surges in heifers after intrauterine infusions of Escherichia coli
endotoxin. American Journal of Veterinary Research 50, p 368–373
28- Quinn PJ, Carter ME, Markey BK, Carter GR (1994), Clinical Veterinary
Microbiology. Wolfe Publishing. PP: 118-143
29- Sagartz JW, Hardenbrook HJ (1971), A clinical, bacteriologic, and histologic survey
of infertile cows. Journal American Veterinary Medicine Association 158, p 619622
30- SAS (1991), SAS for P.C. 6.04. SAS Institute, Inc., Cary, NC, USA
31- Semambo DK, Ayliffe TR, Boyd JS, Taylor DJ (1991), Early abortion in cattle
induced by experimental intrauterine infection with pure cultures of Actinomyces
pyogenes. Veterinary Record 129, p 12–16
32- Sheldon IM (2004), The postpartum uterus. Veterinary Clinics of North America
Food Animal Practice 20, p 569–591
33- Sheldon IM, Dobson H (2004), Postpartum uterine health in cattle. Animal
Reproduction Science 82/83, p 295–306
34- Sheldon IM, Noakes DE, Rycroft AN, Dobson H (2002a), Effect of postpartum
manual examination of the vagina on uterine bacterial contamination in cows.
Veterinary Record 151, p 531–534
-17-
35- Sheldon IM, Noakes DE, Rycroft AN, Pfeiffer DU, Dobson H (2002b), Influence of
uterine bacterial contamination after parturition on ovarian dominant follicle
selection and follicle growth and function in cattle. Reproduction 123, p 837–845
36- Sheldon IM, Noakes DE, Rycroft AN, Dobson H (2003), The effect of intrauterine
administration
of
estradiol
on
postpartum
uterine
involution
in
cattle.
Theriogenology 59, p 1357–1371
37- Sheldon IM, Lewis G, LeBlanc S, Gilbert R (2006), Defining postpartum uterine
disease in dairy cattle. Theriogenology 65, p 1516–1530
38- Studer E, Morrow DA (1978), Postpartum evaluation of bovine reproductive
potential: comparison of findings from genital tract examination per rectum, uterine
culture, and endometrial biopsy. Journal of American Veterinary Medicine
Association 172, p 489-494
39- Trinth HT, Billington SJ, Field AC, Songer JG, Jost BH (2002) Susceptibility of
Arcanobacterium pyogenes from different sources to tetracycline, macrolide and
lincosamide antimicrobial agents. Veterinary Microbiology 85, p 353-359
40- Williams EJ, Fischer DP, Pfeiffer DU, England GC, Noakes DE, Dobson H,
Sheldon IM (2005), Clinical evaluation of postpartum vaginal mucus reflects uterine
bacterial infection and the immune response in cattle. Theriogenology 63, p 102–
117
REPLY FROM AUTHORS
Dear Anna wallentin Searle
Ass. Editor/OJVR/07
Thank you for your consideration of our manuscript.
1- The reviewer comments in text were included to the manuscript.
2- Some questions were answered in the text.
-18-
3- The comments of reviewer on statistics subjects were done.
Structural abnormalities were graded and used either Pearson’s or
Spearman’s Rank testing for strength of correlation.
4- We believe the same as reviewer about additional data to make the
conclusions more robust. In our study 402 postpartum dairy cows
were examined and 89 cows affected with clinical endometritis were
selected, but just 2 E .coli were isolated. According the reports of
other scientists, a wide variety of bacteria can be isolated from
almost all cows during the first 10–14 postpartum days. Bacterial
presence in the uterus is usual at this time and can be detected from
more than 90% of the cows, regardless of disease signs (Sheldon and
Dobson, 2004). The gram-negative bacteria, especially E. coli, seem
to dominate in the uterus within the first days after calving (Hussain
et al., 1990; Huszenicza et al., 1999). The incidence and species of
bacteria gradually decrease along with the pp days. Thus, the
presence of bacteria is sporadic on 28–35 days after calving, and the
uterine cavity should be sterile thereafter (Paisley et al., 1986;
Hussain, 1989; Hussain and Daniel, 1991a,b). Isolation of A.
pyogenes at the late involution period (28–35 days after calving) is
associated
with
dramatically
decreased
re-conception
rate
(Huszenicza et al., 1999). Later after birth, A. pyogenes and gramnegative anaerobic bacteria are found in uterine secretions of animals
suffering from severe puerperal endometritis, especially in
combination with retention of the fetal membranes (Bekana et al.,
1994). The appearance of A. pyogenes seems to be an important
indicator of a puerperal disorder resulting in a disturbed fertility
(Huszenicza et al., 1999). Jadon et al (2005) reported that in cases
dystocia-affected buffaloes among the aerobes, Arcanobacterium
pyogenes was isolated in less frequently before (12.9%) and
immediately after relieving dystocia (12%), than during day’s 24–60
-19-
postpartum (82%). The sample count in our study was similar other
study. So, for additional data, we probably need to examine more
than 1000 cows.
5- Since the qualitative results of the diffusion tests correlate well with
the quantitative results of the dilution tests and the procedure is
simple to perform, the disk diffusion test is widely used throughout
the world. We used the Kirby-Bauer procedure which is the most
common diffusion technique. In this method, the zone diameters of
growth inhibition are expressed as susceptible, intermediate, or
resistant according to an interpretive table. The interpretations for the
antimicrobial agents in such tables are those usually recommended
by the United States Food and Drug Administration and by the
NCCLS. The zones of inhibition are approximately inversely
proportional to the MIC values. The MIC values can be compared to
concentrations of antimicrobial agent achievable at various body
sites as a means of predicting therapeutic efficacy. Taking this into
account, it is possible to extrapolate from zones of inhibition
determined with the disk diffusion methods to a prediction of
therapeutic efficacy.
Microbiologists can only recommend therapeutic agents based
on the results of in vitro susceptibility testing of a bacterial isolate.
However, the practitioner must take the final choice based on his or
her knowledge of all the pertinent facts.
6- The comments of reviewer and new changes are remained in the text
and those are ready to your accept.
Thank you in advance for consideration again
Dr.Mohammad Rahim Ahmadi
Professor of theriogenology
Department of Clinical Science
School of Veterinary Medicine
-20-
Shiraz University
P.O. Box 71345-1731
Shiraz . Iran
References used in above:
Sheldon IM, Dobson H (2004), Postpartum uterine health in cattle. Animal
Reproduction Science 82/83, p 295–306
Paisley, L.G., Mickelsen,W.D., Anderson, P.B., 1986. Mechanisms and therapy for
retained fetal membranes and uterine infections of cows: a review. Theriogenology
25, 353–381.
Hussain, A.M., 1989. Bovine uterine defense mechanisms: a review. J. Vet. Med. B 36,
641–651.
Hussain, A.M., Daniel, R.C.W., 1991a. Bovine normal and abnormal reproductive and
endocrine functions in the postpartum period: a review. Reprod. Domest. Anim. 26,
101–111.
Hussain, A.M., Daniel, R.C.W., 1991b. Bovine endometritis: current and future
alternative therapy. J. Vet. Med. A 38, 641–651.
Huszenicza, Gy., Fodor, M., Gacs, M., Kulcsar, M., Dohmen, M.J.V., Vamos, M.,
Porkolab, L., Kegl, T., Bartyik, J., Lohuis, J.A.C.M., Janosi, Sz., Szita, G., 1999.
Uterine bacteriology, resumption of cyclic ovarian activity and fertility in
postpartum cows kept in large-scale dairy herds. Reprod. Domest. Anim. 34, 237–
245.
Bekana, M., Jonsson, P., Ekman, T., Kindahl, H., 1994. Intra uterine bacterial findings
in postpartum cows with retained fetal membranes. J. Vet. Med. A 41, 663–670.
Jadon, R.S. Dhaliwal, G.S., Jand, S.K. 2005. Prevalence of aerobic and anaerobic
uterine bacteria during peripartum period in normal and dystocia-affected buffaloes.
Animal Reproduction Science 88, 215–224
Carter, G.R. and Cole, Jr. J.R. (1990) Diagnostic Procedures in Veterinary Bacteriology
and Mycology, 5th edition, Academic Press, PP: 479-492.
Overall this paper is well written, well presented and referenced. The majority of
the editorial changes included below are grammatical and intended to help the
authors secure publication. However, I have two major comments on the content,
-21-
which could be rectified via obtaining more field data and adding it to this paper
and modifying the statistaical method.
3.
There is a need for additional data to make the conclusions more robust.

It is not realistic to base a conclusion of antibiotic resistance on 2 E. coli
isolates. There are too few of all other isolates too.

Most isolates were A.pyogenes (18) even so an MIC90 would need an
absolute minimum of 20 isolates. Any statement of antibacterial
resistance even from the simple disk diffusion method (rather than a
titration method) needs to be expressed as percent of resistant isolates
from a meaningful total number of tested isolates.
4. Need to clarify how reproductive tract structural abnormalities are graded
and use a statistical method to correlate these with bacterial isolation. I
suggest the easiest way would be to use a rank system (e.g. 1-4) then use
either Pearson’s or Spearman’s Rank testing for strength of correlation.
If the authors could obtain a substantial additional amount of isolates, use a
statistical method to assess strength of correlation to support their conclusions and
make the suggested grammatical changes this paper could then become suitable
for publication.
-22-
Bacterial study of clinical postpartum endometritis in
Holstein dairy cows
Morteza Yavari1, Masoud Haghkhah2, Mohammad Rahim Ahmadi1
Department of Clinical Sciences1 and Pathobiology2 School of Veterinary Medicine,
Shiraz University, P.O.Box:71345-1731, Shiraz, Iran
a
Corresponding Author:
Mohammad Rahim Ahmadi
Professor of Theriogenology
Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University,
PO Box: 71345-1731, Shiraz, Iran.
Mobile: +98-917-700-1074
Fax: +98-711-228-6950
E-mail: rahmadi@shirazu.ac.ir
-23-
Abstract
Endometritis is inflammation of the endometrial lining of the uterus and is
associated with delayed uterine involution and poor reproductive performance. The aim
of this study was to present the results of bacteriological culture from uterine swabs of
dairy cows affected with postpartum endometritis and to evaluate the antimicrobial
susceptibility of the isolated bacteria. In total, eighty nine Holstein cows affected with
postpartum endometritis were selected and sampled between 21-35 day postpartum.
Swabs (n=89) were collected from the uterine lumen of dairy cattle. Bacteria were
identified following aerobic and anaerobic culture and the disk diffusion method was
used to determine susceptibility of the major pathogenic isolated bacteria. The results
revealed that the most common isolates from cases of endometritis in study cows were
Arcanobacterium pyogenes, E. coli, and non-differentiated streptococci, staphylococci
and bacilli (type?)(we could not understand the opinion of the reviewer about the
type of the bacilli. Indeed bacilli is a plural noun like streptococci and
staphylococci). The antimicrobial susceptibility tests showed that E. coli were sensitive
to enrofloxacin and ceftiofur (only 2 isolated means nothing)-(we explained this
question in the letter to editor), but resistant to tetracycline and oxytetracycline. For
A. pyogenes, 72, 66, 72 and 72 percent of isolates were resistant to oxytetracycline,
tetracycline, enrofloxacin and ceftiofur respectively. All isolates showed resistance to
penicillin. In conclusion, oxytetracycline which is the most traditional antimicrobial
therapy for postpartum endometritis in cows appears not to be efficacious. There is
widespread resistance to enrofloxacin and third generation cephalosporins too.
Therefore, dairy farms need to evaluate alternatives to treating and preventing postpartum endometritis including non antibiotic options.
Keywords: Endometritis; Bacteria; Antimicrobial susceptibility; Uterus; Dairy cow
-24-
Abbreviations:
EPC=Epithelial cells
LVEP=large vacuolated epithelial cells
MAC=macrophage
EN=endometritis
Neut=neutrophils
Lym=lymphocytes
LH= Lutenizing hormone
C=Cervix
Introduction
During parturition, the physical barriers of the cervix, vagina and vulva are
compromised providing the opportunity for bacteria to ascend the genital tract. Bacteria
can be isolated from the uterus of over 90% of cows early postpartum (Griffin et al.,
1974; Paisley et al., 1986). Most healthy cows are able to clear the uterus of bacteria
within the first 2 to 3 weeks after calving (Bondurant, 1999). However, cows that
cannot eliminate the infection may subsequently develop endometritis (Dhaliwal et al.,
2001).
Endometritis is a common reproductive disorder in female domestic animals with
consequences ranging from no effect on reproductive performance to permanent
sterility. It affects the general health of animals and adversely affects their reproductive
performance (Amiridis et al., 2003). Subclinical endometritis, based on uterine
cytological examination, is also prevalent in dairy cows and has a profound negative
impact on reproductive performance (Hammon et al., 2006). The presence of bacteria in
the uterus causes inflammation, histological lesions of the endometrium and delays
uterine involution (Bonnett et al., 1991, Sheldon et al., 2003). In addition, uterine
bacterial infection or bacterial products suppress pituitary LH secretion, and perturb
postpartum ovarian follicle growth and function, which disrupts ovulation in cattle
(Sheldon et al., 2002b, Opsomer et al., 2000, Peter and Bosu., 1988, Peter et al., 1989).
Thus, endometritis is associated with lower conception rates, increased intervals from
calving to first service or conception, and more culls for failure to conceive (Borsberry
and Dobson, 1989, Huszenicza et al., 1999, LeBlanc et al., 2002).
-25-
A variety of antimicrobial agents, administered by intrauterine infusion or
parenteral injection, are used to treat uterine infections (Cohen et al., 1995). There is
little literature available concerning bacterial causes of postpartum endometritis and
their susceptibility to suitable candidate antibiotics in Iranian dairy farms. The aim of
this study was: (a) to identify isolates by bacteriological culture from uterine swabs of
Holstein dairy cows with clinical endometritis between 21 and 35 days postpartum and
(b) to evaluate the antimicrobial susceptibility of the most common isolates.
MATERIALS AND METHODS
Animals
The study was carried out in 13 large commercial dairy herds of Iran. Herds size ranged
from 300 to 1500 cows. Four hundred two postpartum dairy cows were examined once
between 21 and 35 days postpartum. In total 89 cows affected with clinical endometritis
were selected. Cows in all herds were calved in calving boxes hygienically and kept in
individual boxes for at least 10 days after parturition. Corn silage, alfalfa hay, and
concentrates as a total mixed ration were used. None of the cows received any
intrauterine or reproductive hormonal therapy for at least 10 days before sampling for
this study.
Clinical examination
Cows were first examined for the presence of recent discharge on the vulva,
perineum, or tail. If discharge was not visible externally, cows were examined
vaginally. The cow’s vulva was thoroughly cleaned with a dry paper towel and a clean,
lubricated, gloved hand was inserted into the vagina. In each cow, the lateral, dorsal and
ventral walls of the vagina were palpated, and the mucus contents of the vagina
withdrawn manually for examination, as described by Sheldon et al. (2002a). The
vaginal mucus was assessed for color and presence of pus. The nature of the discharge
-26-
was classified as clear mucus, clear mucus with flecks of pus, mucopurulent
(approximately 50% pus and 50% mucus), purulent (>50% pus) but not foul-smelling,
purulent or red-brown and foul smelling using the methodology described by LeBlanc
et al. (2002).
Following vaginal examination, transrectal palpation of the reproductive tract was
performed and cervical diameter, location of the uterus, symmetry of the uterine horns,
diameter of the (larger) uterine horn, texture of uterine wall, palpable uterine lumen,
dominant palpable ovarian status including corpus luteum (CL), follicle, cyst (>2.5 cm
in diameter), or quiescent ovaries was recorded (LeBlanc et al., 2002). (do you mean
palpable abnormalities? – no structure means absence of tract palpated) [structure
word changed to status (follicle>8mm, corpus luteum and some follicle <8mm) no palpable structures changed to quiescent ovaries (ovaries without follicle>8mm
and corpus luteum)].
Ultrasonographic assessment of uterus and ovaries using a 5MHz rectal linear
probe (AMI Company, Canada) was also performed. Diameter of the uterus,
echotexture and thickness of the uterine wall and intraluminal fluid accumulation were
evaluated in the cows. Ovarian structures (follicle, CL and cyst) were scanned and
measured by calipers (Mateus et al., 2002).
Uterine swab collection and bacteriological culture
For each animal, a transcervical guarded swab was collected from the uterine body
(Noakes et al., 1989). The swab comprised a long copper wire bearing a cotton wool tip
sheathed in a metal guard tube (8 mm external diameter; 58 cm long) and was wrapped
and sterilized by autoclaving at 121°C for 15 min. The guard tube was covered by a
sterile plastic sheath to prevent contamination of the swab during the cervix insertion.
After restraining the animal and securing its tail, the perineal region was washed
and cleaned. The cervix was grasped per-rectum and the sterilized catheter was passed
-27-
through the cervix into the uterine body. Then, the inner rod of the catheter was pushed
forward to expose the swab to the endometrium and was rotated against the uterine wall
and then withdrawn within the catheter. To avoid contamination, the catheter was then
cleaned with alcohol. Swabs were cultured immediately on sheep blood agar and
MacConkey agar (MERCK), and incubated at 37°C for 48 h. The same culture on sheep
blood agar (MERCK) was incubated anaerobically for up to 7 days. Standard
biochemical tests were used for the isolation and identification of the isolates as
described by (Quinn et al. (1994).
Blood sampling and progesterone assays
Blood samples were collected from the coccygeal vein or artery into evacuated
tubes and transported on ice to the laboratory. Serum was separated by centrifugation at
2500 rpm for 10 min and stored frozen at -20°C until required. Plasma progesterone
concentration was measured by radioimmunoassay (Spectria® Progesterone RIA,
Espoo, Finland) with a sensitivity of 0.1 ng/ml and intra- and inter-assay coefficients of
variation of 10.2% and 6.5 % respectively.
Antimicrobial susceptibility tests
According to the categorization of bacteria isolated by culture of uterine swabs,
based on their potential pathogenicity within the uterus (Sheldon, 2004),
Arcanobacterium pyogenes and E. coli were the only major pathogens identified and
used for antibiotic susceptibility tests. Disk diffusion method was performed to
determine susceptibility of the major isolated pathogenic bacteria based on the NCCLS
1996 protocol (National Committee for Clinical Laboratory Standards) (Please cite
the reference as NCCLS 1996 etc and add to list at end of paper). The bacterial
suspension turbidity adjusted to McFarland standard number 0.5 in Mueller-Hinton
broth (MERCK) and inoculated on Mueller-Hinton agar (MERCK) with a sterile cotton
swab.
-28-
Commercial antibiotic disks containing single concentrations of each antibiotic
were then placed onto the inoculated plate surface. Inhibition zone of growth around
each disk after overnight incubation at 37°C, were measured. The zone diameter was
interpreted using a zone size interpretation chart (Lorian, 1996). The antibiotics and
their concentration per disc were as follows: tetracycline 30µg, oxytetracycline 30µg,
penicilin 10 international units (iu), enrofloxacin 5µg and ceftiofur 30µg (Quinn et al.,
1994).
Statistical analysis
Data were analyzed by using SAS software, version 6.12.Correlation of ovarian
structure, progesterone serum level and discharge status (is this ranked by severity =
yes) with bacterial culture results in postpartum endometric cows were determined.
Relation or independency of above variables with each other were examined using Chisquare and Fisher exact tests (SAS, 1991).
Results
In total, 89 Holstein cows were selected and sampled at 21-35 day postpartum.
Thirty five (39.3%) swabs were found bacteriologically positive and the remaining 54
(60.7%) showed no bacterial growth. Aerobic and facultative anaerobic bacteria which
were isolated are listed in Table 1. A total of 61 isolates were identified from the
positive swabs. The most frequently isolated facultative anaerobe was A. pyogenes 18
(29.51%) followed by Bacillus spp. 13 (21.31%), Streptococci 8 (13.11%),
Staphylococci (9.83) and Lactobacillus 8 (13.11%). Twenty three (66%) of positive
swabs yielded pure bacterial growth, of which A. pyogenes was the most frequently
isolate (Table 1). Character of uterine discharge and bacterial isolation variables were
dependent and mucus characters had significant different bacterial isolation (P<0.05).
Character of uterine discharge and bacterial isolation variables were dependent and
mucus characters had significant (P<0.05) different bacterial isolation. There was
-29-
significant positive correlation (r=+0.45, P<001) between mucus classification and
bacterial results (Table 2). Ovarian structure (r=-0.14, P>0.05) and P4 level of serum
(r=-0.12, P>0.05) were not correlated with bacterial isolation (Table 3).
The result of antimicrobial susceptibility tests showed that both of E. coli
isolates (not enough to be conclusive)(we explained in editor letter) were sensitive to
enrofloxacin and ceftiofur, but they were resistant to tetracycline and oxytetracycline
(Table 4). For A. pyogenes, (state number of isolates as not many and % is misleadingly
conclusive) 72, 66, 72 and 72 percent of isolates were resistant to oxytetracycline,
tetracycline, enrofloxacin and ceftiofur respectively. All bacteria isolate showed
resistance to penicillin (Fig.1).
TABLE 1. Bacterial isolates from postpartum endometritis of dairy cows
Bacteria
A. pyogenes
E. coli
Streptococci (total)
S. equinus
S. dysgalactiae
S. spp
Staphylococci (total)
S. epidermidis
S. spp
Corynebacterium spp
Mannhiema haemolytica
Micrococcus spp
Bacilli (total)
B. coagulans
B. firmus
B. pumilus
B. spp.
Lactobacillus spp
Total
Number
18
2
8
1
1
6
6
1
5
2
1
3
13
3
3
3
4
8
61
%
29.51
3.28
13.11
1.64
1.64
9.83
9.83
1.64
8.19
3.28
1.64
4.92
21.31
4.92
4.92
4.92
6.56
13.11
100
Table 2. Vaginal mucus discharge and bacterial isolation in postpartum endometritis
cows.
Bacteriology
Clear
mucus
Character of mucus
Mucus with
Mucopurulent
flecks of pus
-30-
Purulent
total
Positive
9(10.11)a 5(5.62)abc
10(11.24)b
11(12.36)c 35(39.33)
Negative
34(38.2)a 11(12.36)abc 7(7.87)b
2(2.25)c
54(60.68)
total
43(48.31) 16(17.98)
17(19.10)
13(14.61) 89(100)
Values with different superscripts in each row are those that differ significantly
(p<0.05)
Table 3. Association of ovarian structure and plasma progesterone concentration with
results of bacterial culture in postpartum endometritis cows
Bacteriology
Positive
Negative
total
Ovarian Structure
Nos. (%)
Static ovaries
Follicle
>8mm
4
1
5 (5.62)
22
35
57 (64.04)
Corpus
luteum
9
18
27 (30.34)
Progesterone
Nos. (%)
< 1ng/ml
> 1ng/ml
26
34
60 (67.4)
9
20
29 (32.6)
(what does negative structure mean?? – static ovaries instead no structures)
do you mean palpable abnormalities? no structure means absence of tract
palpated) – (The word of structure were changed to status. we were used the word
of structure for illustration of ovarian function before.)
Table 4. The antimicrobial susceptibility of the major isolated pathogenic bacteria from
endometritis cows using disk diffusion
Bacteria &
Results
Antibiotics
oxytetracycline
tetracycline
penicillin
ceftiofor
enrofloxacin
Arcanobacterium pyogenes
(number of isolates = 18)
E. coli
(number of isolates = 2!)
Resistance
%
Intermediate
%
Sensitive
%
Resistanc
e%
Intermediat
e%
Sensitive
%
72
66
100
72
72
17
23
0
0
0
11
11
0
28
28
100
0
100
0
0
0
100
0
0
0
0
0
0
100
100
Discussion
The uterine lumen is sterile before parturition and if bacterial invasion occurs,
there is usually resorption of the fetus or abortion (Semambo et al., 1991). During or
shortly after parturition, microorganisms from the animal's environment, skin, and feces
-31-
contaminate the uterine lumen (Sheldon, 2004; Sheldon and Dobson, 2004; Földi et al.,
2006). The flora of the postpartum uterus has been shown to be quite variable, and the
results of one sample may not give a full picture of the infection status (Griffin et al.,
1974). However, intrauterine bacterial infection, does not necessarily present as a
clinical manifestation of disease; this is dependant on the immune status of the host
(Földi et al., 2006).
The results of this study revealed that the most common isolates from cases of
endometritis in cattle were A. pyogenes, streptococci (8) and staphylococci (6) E. coli
(2). (removed this sentence according reviewer comments) In our study, the most
common isolate was A. pyogenes. Studies confirmed that most of the clinical and
reproductive consequences are attributed to the presence of certain non-specific
pathogens: mainly to A. pyogenes, either alone or in combination with other bacteria
such as E. coli and gram negative obligate anaerobes (Földi et al., 2006). Isolation of A.
pyogenes at the late involution period (28-35 days after calving) is associated with
dramatically decreased re-conception rate (Huszenicza et al., 1999). In addition; A.
pyogenes was the most frequent single isolate (40%) in our study. Previous studies have
shown A. pyogenes to be recovered in pure culture more frequently with increased days
postpartum (Hartigan et al., 1974).
Obligate anaerobes such as Fusobacterium necrophorum and Prevotella spp
were not isolated in this study. This result is in agreement with some of other published
reports. Kaczmarowski et al. (2004) reported that anaerobes were of lower significance
in their examinations. In our opinion, it seems that the inconsistency of the bacterial
isolates in different studies may be related to the different bacterial flora of each area
and country. However, Williams et al. (2005) explained that variation between studies
may include the use of clinically ill animals or the selection of animals for
microbiological examination based on likely uterine disease. In addition, the inclusion
-32-
of animals <21 days postpartum in other studies may be important as they have a
different bacterial profile to those calved longer.
The effect of character of vaginal mucus on bacterial isolation was considered
independently in the present study. Positive results from bacterial culture was more
commonly associated with mucopurulent or purulent vaginal mucus (r=+0.45, P<001) .
These observations are in agreement with the previous result of Williams et al. (2005).
They reported that the bacterial growth densities for recognized and potential pathogens
were associated with purulent vaginal mucus, and higher growth density for recognized
pathogens was associated with mucopurulent mucus character. Pus is formed as a result
of bacterial infection by a mixture of viable and dead neutrophil leucocytes, necrotic
tissue and tissue fluid, so it is not surprising that bacterial growth density of pathogenic
bacteria is associated with purulent vaginal mucus (Williams et al., 2005).
It is important to be able to diagnose the presence of uterine infection to
facilitate timely and appropriate treatment and to quantify the severity of disease, which
allows a prognosis to be given for subsequent fertility. Unfortunately, there is no ‘‘gold
standard’’ for diagnosis of uterine disease, making it difficult to measure the sensitivity
and specificity of clinical definitions (Sheldon et al, 2006). Miller et al. (1980)
concluded that vaginoscopic examination is a more accurate method for detecting
uterine infections than palpation per-rectum. In the study by LeBlanc et al. (2002), the
reproductive performance of cows with purulent discharge on vaginoscopic examination
was significantly lower than cows with no abnormal discharge. However, vaginoscopy
has often failed to identify all cows that are truly at risk of poor reproductive
performance (Kasimanickam et al., 2004).
Progesterone suppresses uterine immune defenses and predisposes the uterus to
nonspecific infections. These occur most commonly in postpartum animals and may
reduce the reproductive performance of livestock (Lewis 2004). However, in the present
-33-
study there was no positive association between ovarian structures (r=-0.14, P>0.05)
and progesterone-serum levels (r=-0.12, P>0.05) with bacterial isolation.
According to the categorization of bacteria isolated by culture of uterine swabs,
based on their potential pathogenicity within the uterus (Sheldon, 2004), A. pyogenes
and E. coli were only used for antibiotic susceptibility tests. The results of antimicrobial
susceptibility tests indicated that A. pyogenes
can be resistant to the common
antimicrobial agents (tetracycline, penicillin, enrofloxacin and ceftiofur) used for
intrauterine treatment in the practice field. This is in agreement with previous reports.
Cohen et al. (1995) also found A. pyogenes isolates in the uterus of cows with retained
fetal membranes or postpartum endometritis that were not susceptible to
oxytetracycline.
Other studies have also isolated A. pyogenes that is less susceptible to
antimicrobial agents commonly used as feed and water additives used in the United
State agriculture (i.e. tetracycline, macrolide and lincosamide) (Trinth et al., 2002).
Sheldon et al. (2004) also reported the relative inefficiency of oxytetracycline against A.
pyogenes. However, according to their findings, cephalosporins and the fluroquinolone,
enrofloxacin were effective against all the strains of A. pyogenes tested
In conclusion, use of oxytetracycline, the usual antimicrobial therapy for
postpartum endometritis in Iran, appears not to be efficacious. Dairy farms need to
implement alternative procedures for treatment of postpartum endometritis. According
to the suggestion of Hussain and Daniel (1991), dairy farms may benefit from also
evaluating non antibiotic alternatives for the treatment of postpartum endometritis.
-34-
References
1- Amiridis GS, Fthenakis GC, Dafopoulos J, Papanikolaou T, Vrogianni VS (2003),
Use of cefquinome for prevention and treatment of bovine endometritis. Journal of
Veterinary Pharmacology Therapy 26, p 387–390
2- Bondurant RH (1999), Inflammation in the bovine female reproductive tract. Journal
of Animal Science 77 (Suppl), p 101–110
3- Bonnett BN, Martin SW, Gannon VP, Miller RB, Etherington WG (1991),
Endometrial biopsy in Holstein–Friesian dairy cows. III. Bacteriological analysis
and correlations with histological findings. Canadian Journal of Veterinary
Medicine 55, p 168–173
4- Borsberry S, Dobson H (1989), Periparturient diseases and their effect on
reproductive performance in five dairy herds. Veterinary Record 124, p 217–219
5- Cohen RO, Bernstein M, Ziv G (1995), Isolation and antimicrobial susceptibility of
A. pyogenes recovered from the uterus of dairy cows with retained fetal membranes
and postparturient endometritis. Theriogenology 43, p 1389–1397
6- Dhaliwal GS, Murray RD, Woldehiwet Z (2001), Some aspects of immunology of
the bovine uterus related to treatments for endometritis. Anim Reprod Sci 67:135152
7- Földi J, Kulcsar M, Pecsi A, Huyghe B, de Sa C, Lohuis JA, Cox P, Huszenicza G
(2006), Bacterial complications of postpartum uterine involution in cattle. Animal
Reproduction Science 96, p 265-281
8- Griffin JFT, Hartigan PJ, Nunn WR (1974), Non-specific uterine infection and
bovine fertility. I. Infection patterns and endometritis during the first seven weeks
post-partum. Theriogenology 1, p 91–106
-35-
9- Hammon DS, Evjen IM, Dhiman TR, Goff JP, Walters JL (2006), Neutrophil
function and energy status in Holstein cows with uterine health disorders.
Veterinary Immunology Immunopathology 113, p 21-29
10- Hartigan PJ, Griffin JFI, Nunn WR (1974), Some observations of Corynebacterium
pyogenes infection of the bovine uterus. Theriogenology 1, p 153-167
11- Hussain AM, Daniel RCW (1991), Bovine endometritis: current and future
alternative therapy. Journal of Veterinary Medicine A 38, p 641–651
12- Huszenicza G, Fodor M, Gacs M, Kulcsar M, Dohmen MJW, Vamos M, Porkolab
L, Kegl T, Bartyik J, Lohuis JACM, Janosi Sz, Szita G (1999), Uterine bacteriology,
resumption of cyclic ovarian activity and fertility in postpartum cows kept in largescale dairy herds. Reproduction Domestic Animal 34, p 237–245
13- Kaczmarowski M, Malinowski E, Markiewicz H (2004), Influence of various
treatment methods on bacteriological findings in cows with puerperal endometritis.
Polish Journal of Veterinary Science 7, p 171-174
14- Kasimanickam R, Duffield TF, Foster RA, Gartley CJ, Leslie KE, Walton JS
(2004), Endometrial cytology and ultrasonography for the detection of subclinical
endometritis in postpartum dairy cows. Theriogenology 62, p 9–23
15- LeBlanc S, Leslie K, Duffield T, Bateman K, Keefe GP, Walton JS, Johnson WH
(2002), Defining and diagnosing postpartum clinical endometritis and its impact on
reproductive performance in dairy cows. Journal of Dairy Science 85, p 2223–2236
16- Lewis GS (2004), Steroidal regulation of uterine immune defenses. Animal
Reproduction Science 82/83, p 281–294
17- Lorian V (1996), Antibiotics in Laboratory Medicine. 4th ed. Williams and Wilkins
18- Mateus L, Lopes da Costa L, Bernardo F, Silva JR (2002), Influence of puerperal
uterine infection on uterine involution and postpartum ovarian activity in dairy
cows. Reproduction Domestic Animal 37, p 31–35
-36-
19- Miller HV, Kimsey PB, Kendrick JW, Darien B, Doering L, Franti C (1980),
Endometritis of dairy cattle: diagnosis, treatment and fertility. Bovine Practitioner 5,
p 13–23
20- National Committee for Clinical Laboratory Standards. Performans Standards for
Antimicrobial Disk Susceptibility Test. 6th ed. Approved Standard M2-A6, Vol 17
No. 1, Villonova, PA: NCCLS, 1996.
21- Noakes DE, Till D, Smith GR (1989), Bovine uterine flora post partum: a
comparison of swabbing and biopsy. Veterinary Record 124, p 563–564
22- Opsomer G, Grohn YT, Hertl J, Coryn M, Deluyker H, de Kruif A (2000), Risk
factors for post partum ovarian dysfunction in high producing dairy cows in
Belgium: a field study. Theriogenology 53, p 841–857
23- Paisley LG, Mickelson WD, Anderson PB (1986), Mechanisms and therapy for
retained fetal membranes and uterine infections of cows: a review. Theriogenology
3, p 353-381
24- Peter AT, Bosu WTK (1988), Relationship of uterine infections and folliculogenesis
in dairy cows during early puerperium. Theriogenology 30, p 1045–1051
25- Peter AT, Bosu WTK, DeDecker RJ (1989), Suppression of preovulatory
luteinizing hormone surges in heifers after intrauterine infusions of Escherichia coli
endotoxin. American Journal of Veterinary Research 50, p 368–373
26- Quinn PJ, Carter ME, Markey BK, Carter GR (1994), Clinical Veterinary
Microbiology. Wolfe Publishing. PP: 118-143
27- SAS (1991), SAS for P.C. 6.04. SAS Institute, Inc., Cary, NC, USA
28- Semambo DK, Ayliffe TR, Boyd JS, Taylor DJ (1991), Early abortion in cattle
induced by experimental intrauterine infection with pure cultures of Actinomyces
pyogenes. Veterinary Record 129, p 12–16
-37-
29- Sheldon IM (2004), The postpartum uterus. Veterinary Clinics of North America
Food Animal Practice 20, p 569–591
30- Sheldon IM, Dobson H (2004), Postpartum uterine health in cattle. Animal
Reproduction Science 82/83, p 295–306
31- Sheldon IM, Noakes DE, Rycroft AN, Dobson H (2002a), Effect of postpartum
manual examination of the vagina on uterine bacterial contamination in cows.
Veterinary Record 151, p 531–534
32- Sheldon IM, Noakes DE, Rycroft AN, Pfeiffer DU, Dobson H (2002b), Influence of
uterine bacterial contamination after parturition on ovarian dominant follicle
selection and follicle growth and function in cattle. Reproduction 123, p 837–845
33- Sheldon IM, Noakes DE, Rycroft AN, Dobson H (2003), The effect of intrauterine
administration
of
estradiol
on
postpartum
uterine
involution
in
cattle.
Theriogenology 59, p 1357–1371
34- Sheldon IM, Lewis G, LeBlanc S, Gilbert R (2006), Defining postpartum uterine
disease in dairy cattle. Theriogenology 65, p 1516–1530
35- Studer E, Morrow DA (1978), Postpartum evaluation of bovine reproductive
potential: comparison of findings from genital tract examination per rectum, uterine
culture, and endometrial biopsy. Journal of American Veterinary Medicine
Association 172, p 489-494
36- Trinth HT, Billington SJ, Field AC, Songer JG, Jost BH (2002) Susceptibility of
Arcanobacterium pyogenes from different sources to tetracycline, macrolide and
lincosamide antimicrobial agents. Veterinary Microbiology 85, p 353-359
37- Williams EJ, Fischer DP, Pfeiffer DU, England GC, Noakes DE, Dobson H,
Sheldon IM (2005), Clinical evaluation of postpartum vaginal mucus reflects uterine
bacterial infection and the immune response in cattle. Theriogenology 63, p 102–
117
-38-
-39-