- Email: [email protected]
Bacterial invasion of the uterus and oviducts in bovine pyometra
Accepted Manuscript Bacterial invasion of the uterus and oviducts in bovine pyometra C.C. Karstrup, H.G. Pedersen, T.K. Jensen, J.S. Agerholm PII:
S0093-691X(17)30039-0
DOI:
10.1016/j.theriogenology.2017.01.027
Reference:
THE 13982
To appear in:
Theriogenology
Received Date: 1 November 2016 Revised Date:
13 January 2017
Accepted Date: 15 January 2017
Please cite this article as: Karstrup CC, Pedersen HG, Jensen TK, Agerholm JS, Bacterial invasion of the uterus and oviducts in bovine pyometra, Theriogenology (2017), doi: 10.1016/ j.theriogenology.2017.01.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Revised
1
Bacterial invasion of the uterus and oviducts in bovine pyometra
2
C. C. Karstrupa, H. G. Pedersena, T. K. Jensenb, J. S. Agerholma,1.
3 4
a
5 6
b
RI PT
Department of Large Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Dyrlægevej 68, DK – 1870 Frederiksberg, Denmark National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, DK – 1870 Frederiksberg, Denmark
7
Keywords: Domain Bacteria, Fusobacterium necrophorum, Porphyromonas levii, Trueperella pyogenes, Cow, Fluorescence in situ hybridization.
SC
8 9 10
Abstract
12 13 14 15 16 17 18 19 20 21 22 23
Pyometra is a common disease of cattle that causes infertility and thereby financial losses to the cattle industry. Bacteria involved in the development and progression of pyometra have been investigated by microbial culture but their tissue invading abilities, which is an important aspect of bacterial pathogenicity and development of lesions, have not been investigated. Bacterial invasion of the uterus and oviducts was studied in 21 cows diagnosed with pyometra at the time of slaughter by applying fluorescence in situ hybridization using probes targeting 16S ribosomal RNA of Fusobacterium necrophorum, Porphyromonas levii, Trueperella pyogenes and the overall bacterial domain Bacteria. Fusobacterium necrophorum and P. levii were found to invade the endometrium, especially if the endometrium was ulcerated, and penetrated deep into the lamina propria. These species co-localized within the tissue thus indicating a synergism. Trueperella pyogenes did not invade the uterine tissue. In addition to endometrial lesions, most cows with pyometra also had salpingitis but without significant bacterial invasion of the oviductal wall.
TE D
M AN U
11
25 26
AC C
EP
24
27
1
28
E-mail address: [email protected] (J.S. Agerholm)
Corresponding author Tel.: +4529178136
1
ACCEPTED MANUSCRIPT Revised
1. Introduction
30 31 32 33 34 35
Endometrial bacterial contamination develops in up to 90% of cows after parturition [1]. Bacterial contamination can progress into a postpartum infection, leading to conditions such as endometritis, metritis, and pyometra [2]. The diseases are associated with reduced reproductive performance, reduced milk production, milk withdrawal and increased costs for treatment leading to economic losses [3–12]. The annual costs of bovine postpartum uterine infections were estimated to be around €1.4 billion in the EU and around $650 million in the United States in 2009 [12].
36 37 38 39 40 41 42 43 44 45 46
Pyometra accounts for 2% to 5% of the clinical cases of uterine diseases in cows [13,14] and is characterized by inflammation of the endometrium and accumulation of a purulent exudate in the uterine lumen in the presence of a persistent corpus luteum and a closed cervical canal [15,16]. Cows that ovulate early in the postpartum period, when bacterial contamination is still present in the uterus, are at higher risk of developing pyometra [17]. If endometritis is present after ovulation and during diestrus, the inflammation may inhibit the endometrial prostaglandin production thus preventing corpus luteum regression [16,17]. This inhibition causes a permanent closure of the cervical canal and subsequent accumulation of pus in the uterine lumen [16,17]. Affected cows show no systemic signs of illness, but are infertile [18]. Pyometra is usually treated by injection of PGF2α, which induces regression of the corpus luteum leading to relaxation of the cervix and expulsion of the intrauterine exudate with subsequent estrus as a result [19].
SC
M AN U
EP
TE D
The bacterial flora in cases of bovine pyometra has been determined using conventional culture methods [17,20–23] and recently by culture-independent methods [24]. The most frequently isolated bacteria cultured from cases of pyometra are Trueperella pyogenes and Gram-negative bacteria [20,21], especially Fusobacterium necrophorum [17]. In addition to these frequently isolated bacteria, Escherichia coli, Bacteroides melaninogenicus, streptococci, staphylococci, Pasteurella haemolytica, Bacillus spp. and Diphtheroides spp. have been cultured [17,21]. The central role of T. pyogenes has been shown experimentally by inducing pyometra in postpartum cows either by inoculation of a monoculture or together with F. necrophorum and B. melaninogenicus [22,23]. By using next generation sequencing, we found bacteria belonging to the families Fusobacteriaceae (includes F. necrophorum), Mycoplasmataceae, Bacteroidaceae, Porphyromonadaceae and Pasteurellaceae in cases of bovine pyometra with Fusobacteriaceae as the most frequently found bacterium [24]. Actinomycetaceae and Enterobacteriaceae, which include T. pyogenes and E. coli, respectively, were however only a minor part of the operational taxonomic units [24].
AC C
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69
RI PT
29
Culturing and sequencing methods are useful to provide information on bacteria present in cases of pyometra. We have previously applied bacterial sequence analysis to the luminal content and endometrium of cows with pyometra [24], while other studies have focused only on the luminal content [17,20,24] and therefore not provided insight into the invasive properties of the bacteria present. Assessment of invasive properties is important as invasiveness may be associated with development of chronic endometrial lesions influencing the prognosis for fertility. Furthermore, treatment with antibiotics after restoration of cyclicity by PGF2α may be more effective if targeting tissue invading bacteria.
2
ACCEPTED MANUSCRIPT Revised
70 71 72
Therefore, we used fluorescence in situ hybridization (FISH) to study the localization of bacteria in the stroma of the uterus and oviducts in cases of bovine pyometra and to determine to which extent F. necrophorum, T. pyogenes and Porphyromonas levii invade these structures.
RI PT
73
2. Materials and Methods
75 76 77 78 79 80 81 82
2.1 Animals Reproductive tracts of dairy and beef cows with pyometra were collected at a slaughterhouse and sent to the University of Copenhagen for examination from August 2012 to August 2013. Cooling was initiated at the slaughterhouse shortly after slaughter and the organs were kept at 4 ºC during storage and transportation. Inclusion criteria were: 1) the cow was slaughtered more than 21 days postpartum, 2) the uterus was distended with pus, 3) the cervical canal was closed and 4) a corpus luteum was present in one or both ovaries.
83
2.2 Sampling
84 85 86 87 88 89
Before opening the uterus, the serosal surface was wiped with 70% ethanol and flame sterilized at the incision point. A full thickness specimen of about 1x1 cm was taken from the dorsal aspect of the base of the left uterine horn using sterile forceps and a scalpel. The uterine sample and the entire oviducts were fixed in 10% neutral buffered formalin. An endometrial tissue sample (0.5 x 0.5 cm) and 1 mL of luminal exudate were also sampled and used for 16S rRNA polymerase chain reaction combined with next generation sequencing. These results have been published separately [24].
TE D
90
M AN U
SC
74
2.2.1 Histology
92 93 94 95
The formalin fixed specimens were trimmed and from the oviducts, the ampullae were isolated. The tissues were processed by routine methods, embedded in paraffin, sectioned at 3 µm and stained with hematoxylin and eosin. Selected specimens were also stained by Luna’s method for eosinophilic granula.
EP
91
97 98 99 100 101 102
AC C
96
2.2.2 FISH
Sections for FISH were mounted on SuperFrost®Plus slides (Menzel-Gläser, Germany), and 16S rRNA oligonucleotide probes targeting F. necrophorum, P. levii, T. pyogenes and the overall bacterial domain Bacteria (Eub-338) were used (Table 1). Positive and negative controls were included in each hybridization. Positive controls consisted of sections containing the specific bacterial species and processed with the respective probes, while negative controls were not processed with the probe.
103
3
ACCEPTED MANUSCRIPT Revised
The hybridizations were performed in Shandon hybridization racks (Thermo Scientific, UK), using 99 µL in situ hybridization buffer (1 M Tris (pH 7.2), 5 M NaCl, 10% SDS, H2O) added 1 µL probe. The slides were incubated at 46 °C for 16 to 24 h, rinsed 3 x 3 min with in situ hybridization buffer and 3 x 3 min with in situ washing solution (1 M Tris (pH 7.2), 5 M NaCl, H2O), and finally dipped in MilliQ water 10 to 15 times at room temperature.
109 110 111 112 113 114
The slides were mounted with Vectashield® (Vector Laboratories, INC. Burlingame, CA, USA), and evaluated using an Axiomager M1 epifluorescence microscope with a 100-W HBO lamp. Filter sets 43 and 38 were used to visualize Cy3 (excitation/emission peak at 552/570 nm) and FITC (excitation/emission peak at 495/520 nm), respectively. Images were obtained using an AxioCam MRm version 3 FireWiremonocrome camera and AxioVision software, version 4.5 (Carl Zeiss, Oberkochen, Germany).
115 116 117
The slides were evaluated by one examiner (CCK) on the day of hybridization at X 400 magnification. The presence and the specific localization of bacteria within the tissue or on the surface of the mucosa were recorded.
M AN U
SC
RI PT
104 105 106 107 108
118 119
2.3 Data management
120
Calving date, date of slaughter and parity were extracted from the Danish Cattle Data Base.
121
3. Results
123
3.1 Study population
124 125 126 127 128 129
Twenty-one cows were included in the study. Twenty reproductive tracts were examined within 48 h of slaughter whereas one tract was examined 96 h after slaughter. The left oviduct and ovary from one cow was lost during slaughter. The parity ranged from one to six, with a mean parity of 2.9. The cows were on average 290 days after parturition [range 26 to 1093 days]. One cow had been treated for endometritis (76 days before slaughter) and three others for retained fetal membranes (100, 112 and 462 days before slaughter). Details on treatment were not available.
EP
AC C
130
TE D
122
131
3.2 Histology
132
3.2.1 Uterus
133 134 135 136 137 138
Most of the specimens had a single layer of columnar epithelium through which neutrophils migrated. Endometrial polyps (Fig. 1A) were present in 38.1% (8/21) of the specimens, and 47.6% (10/21) had endometrial ulceration characterized by loss of epithelium, deposits of fibrin and massive infiltration with neutrophils and mononuclear cells in the adjacent lamina propria. Ulceration with development of granulation tissue (Fig. 1B) was present in 19.0% (4/21) of the specimens. These ulcerations were characterized by well-vasculized connective tissue hyperplasia (fibrosis, granulation tissue), which was
4
ACCEPTED MANUSCRIPT Revised
covered by fibrin. In cases without a single layer of columnar epithelium, the epithelium was either missing due to extensive ulceration or the epithelium was altered to a pseudostratisfied columnar epithelium.
142 143
The thickness of the lamina propria was generally normal but was atrophic in five cases (Fig. 1C) where endometrial fibrosis, non-suppurative inflammation and atrophy of glands were present.
144 145 146 147 148
The lamina propria was infiltrated by mononuclear cells and neutrophils in most cases (19/21). Infiltration was often diffuse and affected the upper 25% to 50% of the lamina propria, but in four cases the inflammation involved the entire lamina propria. Nodular lymphoid hyperplasia (Fig. 1C) was present in 61.9% (13/21) of the specimens. Two specimens did not have inflammation of the lamina propria at all.
149 150 151 152 153
Endometrial glands were evenly distributed over the entire specimen in 17 cases, while 3 cases had a multifocal distribution and in one case glands were completely absent. The glands were coiled and covered by tall columnar epithelial cells, except for one case where the glandular epithelium was flattened. Periglandular fibrosis (Fig. 1C) was observed in 47.6% (10/21) of the specimens. Tunica muscularis and the serosa were not affected in any of the specimens.
M AN U
SC
RI PT
139 140 141
154
3.2.2 Oviducts
156 157 158 159 160 161
A folded luminal surface with a single layer of intact ciliated epithelium was present in all but one oviduct. Either uni- or bilateral ampullar salpingitis was present in 18 cows (Fig. 1D). The inflammation was characterized by a luminal debris containing bacteria (Fig 2B) and cellular infiltration of the superficial part of the lamina propria, mainly by lymphocytes and eosinophils. Scattered neutrophils were present and occasionally macrophages were observed. Nodular lymphoid hyperplasia was present in 9.8% (4/41) of the oviducts.
162 163 164
One oviduct from one cow differed markedly from the others. This oviduct was distended to a diameter about six times greater than usually observed and no folds were present. The epithelium was ulcerated and glands were present in the tunica muscularis (adenomyosis).
165
AC C
EP
TE D
155
166
3.3 FISH
167 168 169
Initially, the positive and negative controls were examined. For each section the borders were examined for presence of bacteria to exclude contamination of the individual section. Contamination was not observed.
170 171 172 173 174
3.3.1. Uterus By using the general bacterial probe Eub-338, bacteria were found on the endometrial surface, in the luminal debris and in the lamina propria (Fig. 2A). Bacteria were present in the lumen of the uterus in all cows and within the endometrium in 42.9% (9/21) of the cows (Table 2).
5
ACCEPTED MANUSCRIPT Revised
Fusobacterium necrophorum and T. pyogenes were both found in 76.2% (16/21) of the cows. While F. necrophorum was tissue invasive in 56.3% (9/16) of the cases, T. pyogenes was predominantly (87.5%; 14/16) found in the lumen or on the endometrial surface. Porphyromonas levii was less prevalent (33.3%) but when present, this bacterium had invaded the endometrium in all cases (Table 2). Bacterial invasion of the lamina propria was only observed in relation to endometrial ulcers. While T. pyogenes only invaded the outermost part of the lamina propria, F. necrophorum and P. levii penetrated deep into the lamina propria. Examination for co-localization of the bacteria revealed that F. necrophorum and T. pyogenes did not co-localize in the tissue, but P. levii was consistently located close to F. necrophorum.
185
3.3.2 Oviducts
186 187 188 189 190 191 192 193
Bacteria were present in at least one oviduct in 76.2% (16/21) of the cows when the general Eub-338 probe was applied. Of these, seven cows (43.8%) had bacteria in the ampulla unilaterally and nine cows (56.2%) had bilateral infection. The bacteria were primarily located on the epithelial surface and in the luminal debris (Table 3; Fig. 2B). Fusobacterium necrophorum, P. levii and T. pyogenes were found in the outermost part of the lamina propria in only one case with ulceration of the ampulla. Otherwise the bacteria were highly prevalent intraluminally (Table 3).
194
4. Discussion
195 196 197 198 199 200 201 202 203
FISH examination of uterine tissue samples revealed that F. necrophorum and T. pyogenes were present in most cases (76.2%), but their location differed as T. pyogenes was predominantly present in the uterine lumen or on the endometrial surface while F. necrophorum was tissue invasive in approximately half of the cases. Porphyromonas levii was less prevalent (33.3%), but when present this bacterium had invaded the tissue in all cases. Knudsen and coworkers [24], who examined endometrial specimens of the same cows, observed that the highest number of operational taxonomic units belonged to the family Fusobacteriaceae (including F. necrophorum) in accordance with the FISH results of the present study. Also, culture based studies have shown that F. necrophorum is frequently present in cases of bovine pyometra [17,20,21].
205 206 207 208 209 210 211 212
SC
M AN U
TE D
EP
AC C
204
RI PT
175 176 177 178 179 180 181 182 183 184
Porphyromonas levii has not previously been isolated from the uterus of cows with pyometra, but we included this species in the present study as it often co-localized with F. necrophorum in bovine postpartum endometritis (unpublished results) and because bacterial sequencing found a large number of reads from the family Porphyromonadaceae, which includes P. levii [24]. In addition to F. necrophorum and T. pyogenes, a wide range of other bacterial species have also been identified in culture based studies [17,20,21]. Similarly, bacteria other than F. necrophorum, P. levii and T. pyogenes, i.e. bacteria only identified by using the Eub-338 probe, accounted for approximately 2/3 of the overall bacterial load in the uterine specimens in the present study.
6
ACCEPTED MANUSCRIPT Revised
Culture-based studies of bovine pyometra have been performed on the uterine luminal content [17,20,21]. However, in this study, we focused on the tissue invading bacteria. Among the pathogens investigated, only F. necrophorum and P. levii had invaded the endometrium while T. pyogenes was mainly present in the lumen or on the endometrial surface. When comparing the results from applying the general bacterial probe (Eub-338) and the specific probes for F. necrophorum and P. levii, these latter species were found to be the only, or the absolutely dominating, bacterial species penetrating deep into the lamina propria, thus emphasizing their role in causing tissue damage in bovine pyometra. The co-localization of these species indicates a synergism that may exacerbate their pathogenicity. Such synergism has also been reported by others [25,26]. The tissue invading properties may be of importance as the extent of tissue damage likely will affect the cow’s ability to become pregnant when reproductive cyclicity is restored after treatment with PGF2α. Treatment with a combination of PGF2α and either oxytetracycline or cephapirin has been shown to increase pregnancy rates compared to nontreated cows [27]. Trueperella pyogenes was not found within the endometrium and thus did not show significant tissue invading properties, but the bacterium may play a role in the development and progression of pyometra through production of exotoxins such as pyolysin [28,29]. The invasive properties and co-localization of F. necrophorum and P. levii and the luminal localization of T. pyogenes found in this study are similar to findings in a study of bovine postpartum endometritis (unpublished results).
231 232 233 234 235 236 237
The presence of bacteria and inflammation in oviducts from cows with pyometra has not been reported previously, although damage to the oviducts is likely to influence the subsequent fertility even if cyclicity is regained. We found uni- or bilateral bacterial infection or salpingitis as detected by histology in 76.2% and 69.2% of the examined cows, respectively. Bacterial infection of the oviducts and salpingitis may therefore be common in bovine pyometra. However, lesions were generally less severe in the oviducts than in the uterus and restoration of a normal function may be possible if a normal uterine function and environment is achieved.
238
TE D
M AN U
SC
RI PT
213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230
5. Conclusions
240 241 242 243 244
In the present study we showed that F. necrophorum and P. levii penetrate deeply into the endometrium in bovine pyometra, especially if the endometrium is ulcerated. Also, these bacterial species seem to display a synergism as they co-localize in the tissue. Trueperella pyogenes does not display significant tissue invading properties but may participate in the development and progression of pyometra by other mechanisms. In addition to endometrial lesions, most cows with pyometra develop salpingitis.
AC C
245
EP
239
246
Acknowledgement
247 248 249
We thank the staff at DC Tønder for participating in this study by submitting materials of slaughtered cows. The technical assistance of Mrs. Annie Ravn Pedersen, Mrs. Heidi Holm and Mrs. Anne Dorte Roed is greatly acknowledged.
250
7
ACCEPTED MANUSCRIPT Revised
251
Funding
252 253
This study was performed as part of a PhD program funded by the University of Copenhagen and financially supported by Mælkeafgiftsfonden.
255
Competing interests
256
The authors declare that they have no competing interests.
257
RI PT
254
References
259 260 261
[1]
Griffin JFT, Hartigan PJ, Nunn WR. Non-specific uterine infection and bovine fertility I. Infection patterns and endometritis during the first seven weeks post-partum. Theriogenology 1974;1:91–106.
262
[2]
Sheldon IM. Post-partum uterine infections and their treatment. Cattle Pract 1999;7:23–4.
263 264 265
[3]
Salasel B, Mokhtari A, Taktaz T. Prevalence, risk factors for and impact of subclinical endometritis in repeat breeder dairy cows. Theriogenology 2010;74:1271–8. doi:10.1016/j.theriogenology.2010.05.033.
266 267 268
[4]
Leblanc SJ, Duffield TF, Leslie KE, Bateman KG, Keefe GP, Walton JS, et al. Defining and diagnosing postpartum clinical endometritis and its impact on reproductive performance in dairy cows. J Dairy Sci 2002;85:2223–36. doi:10.3168/jds.S0022-0302(02)74302-6.
269 270 271
[5]
Williams EJ, Fischer DP, Noakes DE, England GCW, Rycroft A, Dobson H, et al. The relationship between uterine pathogen growth density and ovarian function in the postpartum dairy cow. Theriogenology 2007;68:549–59. doi:10.1016/j.theriogenology.2007.04.056.
272 273
[6]
Sheldon M, Noakes DE, Rycroft AN, Dobson H, Sheldon IM. Effect of postpartum manual examination of the vagina on uterine bacterial contamination in cows. Vet Rec 2002;151:531–4.
274 275
[7]
Barlund CS, Carruthers TD, Waldner CL, Palmer CW. A comparison of diagnostic techniques for postpartum endometritis in dairy cows. Theriogenology 2008;69:714–23.
276 277
[8]
Hammon DS, Holyoak GR, Jenson J, Bingham HR. Effects of endometritis at the begining of the breeding period on reproductive performance in dairy cows. AABP Proc 2001;34:142–3.
278 279 280
[9]
Studer E, Morrow Da. Postpartum evaluation of bovine reproductive potential: comparison of findings from genital tract examination per rectum, uterine culture, and endometrial biopsy. J Am Vet Med Assoc 1978;15172:489–94.
281 282 283
[10]
Gilbert RO, Shin ST, Guard CL, Erb HN, Frajblat M. Prevalence of endometritis and its effects on reproductive performance of dairy cows. Theriogenology 2005;64:1879–88. doi:10.1016/j.theriogenology.2005.04.022.
284 285
[11]
Borsberry S, Dobson H. Periparturient diseases and their effect on reproductive performance in five dairy herds. Vet Rec 1989;124:217–9.
286
[12]
Sheldon IM, Cronin J, Goetze L, Donofrio G, Schuberth H-J. Defining postpartum uterine
AC C
EP
TE D
M AN U
SC
258
8
ACCEPTED MANUSCRIPT Revised
disease and the mechanisms of infection and immunity in the female reproductive tract in cattle. Biol Reprod 2009;81:1025–32. doi:10.1095/biolreprod.109.077370.
287 288
[13]
Mickelsen WD, Paisley LG, Anderson PB. Survey of the prevalence and types of infertility in beef cows and heifers. J Am Vet Med Assoc 1986;1:51–4.
291 292
[14]
Sheldon IM, Williams EJ, Miller ANA, Nash DM, Herath S. Uterine diseases in cattle after parturition. Vet J 2008;176:115–21. doi:10.1016/j.tvjl.2007.12.031.
293 294
[15]
Sheldon IM, Lewis GS, LeBlanc S, Gilbert RO. Defining postpartum uterine disease in cattle. Theriogenology 2006;65:1516–30. doi:10.1016/j.theriogenology.2005.08.021.
295 296
[16]
Bondurant RH. Inflammation in the bovine female reproductive tract. J Anim Sci 1999;77:101– 10.
297 298 299
[17]
Olson JD, Ball L, Mortimera RG, Farin PW, Adney WS, Huffman EM. Aspects of bacteriology and endocrinology of cows with pyometra and retained fetal membranes. Am J Vet Res 1984;45:2251–5.
300 301
[18]
Palmer C. Postpartum uterine infection. Bov. Reprod., 2014, p. 440–8. doi:10.1002/9781118833971.ch50.
302 303
[19]
Gustafsson BK, Bäckström G, Edqvist LE. Treatment of bovine pyometra with prostaglandins: An evaluation of a field study. Theriogenology 1976;6:45–50.
304 305
[20]
Noakes DE, Wallace LM, Smith GR. Pyometra in a Friesian heifer: Bacteriological and endometrial changes. Vet Rec 1990;126:509.
306 307 308
[21]
Brodzki P, Kostro K, Brodzki A, Niemczuk K, Lisiecka U. Cytometric analysis of surface molecules of leucocytes and phagocytic activity of granulocytes and monocytes/macrophages in cows with pyometra. Reprod Domest Anim 2014;49:858–64. doi:10.1111/rda.12381.
309 310 311
[22]
Davidson J a, Wright DJ, Archbald LF, Klapstein E, Gottshall SL, Hansen PJ. Effect of induced pyometra on luteal lifespan and uterine fluid concentrations of prostaglandins and interferons in cows. Theriogenology 1996;45:459–70. doi:10.1016/0093-691X(95)00382-I.
312 313 314
[23]
Farin PW, Ball L, Olson JD, Mortimer RG, Jones RL, Adney WS, et al. Effect of Actinomyces pyogenes and Gram-negative anaerobic bacteria on the development of bovine pyometra. Theriogenology 1989;31:979–89. doi:10.1016/0093-691X(89)90481-0.
315 316 317
[24]
Knudsen LRV, Karstrup CC, Pedersen HG, Agerholm JS, Jensen TK, Klitgaard K. Revisiting bovine pyometra - New insights into the disease using a culture-independent deep sequencing approach. Vet Microbiol 2015;175:319–24. doi:10.1016/j.vetmic.2014.12.006.
318 319
[25]
Kan IY, Blum S, Elad D. Synergism between Porphyromonas levii and Arcanobacterium pyogenes in a murine abscess model. Isr J Vet Med 2009;64: En62-En65, He23-24.
320 321
[26]
Bekana M, Jonsson P, Ekman T, Kindahl H. Intrauterine bacterial findings in postpartum cows with retained fetal membranes. J Vet Med Ser A 1994;41:663–70.
322 323 324
[27]
El-Tahawy AEGS, Fahmy MM. Partial budgeting assessment of the treatment of pyometra, follicular cysts and ovarian inactivity causing postpartum anoestrus in dairy cattle. Res Vet Sci 2011;90:44–50. doi:10.1016/j.rvsc.2010.04.017.
325
[28]
Jost BH, Billington SJ. Arcanobacterium pyogenes: molecular pathogenesis of an animal
AC C
EP
TE D
M AN U
SC
RI PT
289 290
9
ACCEPTED MANUSCRIPT Revised
opportunist. Int J Gen Mol Microbiol 2005;88:87–102. doi:10.1007/s10482-005-2316-5.
326 327 328 329
[29]
Amos MR, Healey GD, Goldstone RJ, Mahan SM, Düvel A, Schuberth H-J, et al. Differential endometrial cell sensitivity to a cholesterol-dependent cytolysin links Trueperella pyogenes to uterine disease in cattle. Biol Reprod 2014;90:54. doi:10.1095/biolreprod.113.115972.
RI PT
330
AC C
EP
TE D
M AN U
SC
331
10
ACCEPTED MANUSCRIPT Revised
1
Tables
2
4 5
Table 1: Oligonucleotide probes* used for locating bacteria in uterine and oviductal tissue in cows with pyometra. Name of the probe
Target sequence (5´-3´)
Domain Bacteria Fusobacterium necrophorum Porphyromonas levii Trueperella pyogenes
S-D-Eub-338 S-S-F.necrop-183 P.levii-443 S-S-A.pyogenes-a-A
5´ gctgcctcccgtaggagt 3´ 5´ gattcctccatgcgaaaa 3´ 5´ tacctacgtttactcgcc 3´ 5´ gcacataccgtcacaaaa 3´
M AN U
*Eurofins MWG Operon, Ebersberg, Germany.
7 8 9
Table 2: Location of bacteria in the uterus of cows with pyometra (n = 21) determined by fluorescence in situ hybridization (FISH) using probes for Fusobacterium necrophorum, Porphyromonas levii, Trueperella pyogenes and the overall bacterial domain Bacteria.
TE D
10 11 12
Target Fluorochromes References region 16s FITC [25,26] 16s FITC [26] 16s Cy3 [27] 16s FITC
SC
Target bacteria
6
RI PT
3
14
AC C
13
Localization of bacteria Both luminally* Only Only in the lamina and in the lamina luminally* propria propria Domain Bacteria 100.0% (21/21) 42.9% (9/21) 57.1% (12/21) 0% F. necrophorum 76.2% (16/21) 56.3% (9/16) 43.8% (7/16) 0% P. levii 33.3% (7/21) 100.0% (7/7) 0.0% (0/7) 0% T. pyogenes 76.2% (16/21) 12.5% (2/16) 87.5% (14/16) 0% *either in luminal debris or on the endometrial surface
EP
FISH positive cows
1
ACCEPTED MANUSCRIPT Revised
15
Table 3: Location of bacteria in the oviductal ampulla of cows with pyometra (n = 21) determined by fluorescence in situ hybridization (FISH) using probes for Fusobacterium necrophorum, Porphyromonas levii, Trueperella pyogenes and the overall bacterial domain Bacteria.
RI PT
16 17 18
FISH positive cows (Uni- or bilaterally)
20
SC
Only in the lamina propria 0% 0% 0% 0%
Figure legends
21 22
EP
TE D
Figure 1. Photomicrographs of lesions in the endometrium and oviducts of cows with pyometra. A. An endometrial polyp (P) protrudes into the uterine lumen. The polyp is covered by a pseudostratisfied columnar epithelium and the lamina propria is heavily infiltrated with mononuclear inflammatory cells (asterisk). B. Chronic endometritis characterized by presence of inflamed granulation tissue (G) covered by fibrin (arrowheads) and infiltration with neutrophils (arrows). C. Chronic endometritis characterized by diffuse endometrial atrophy, fibrosis (F) and focal accumulation of lymphocytes (asterisk). Glands are absent except for a single gland with periglandular fibrosis (arrow). D. Suppurative inflammation of the oviduct. Numerous neutrophils migrate through the epithelium (arrowheads) and accumulate in the lumen (arrows). A-D: Hematoxylin and eosin.
AC C
23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Both luminally* Only luminally* and in the lamina propria 12.5% (2/16) 87.5% (14/16) 8.3% (1/12) 91.7% (11/12) 14.3% (1/7) 85.7% (6/7) 6.3% (1/16) 93.7% (15/16)
M AN U
19
Domain Bacteria 76.2% (16/21) F. necrophorum 81.3% (13/16) P. levii 100.0% (7/7) T. pyogenes 68.8% (11/16) *either in luminal debris or on the mucosa
Localization of bacteria
Figure 2. Fluorescence in situ hybridization photomicrographs
A. Endometrial ulceration with numerous bacteria (green fluorescence) invading the tissue. General bacteria probe (Domain Bacteria, Eub-338). Yellow fluorescence represents autofluorescence omitted by erythrocytes. B. Fusobacterium necrophorum (green fluorescence) and Porphyromonas levii (red fluorescence) located in the lumen of the oviduct. C. Trueperella pyogenes (red fluorescence) and other bacteria (Domain Bacteria, Eub-338; green fluorescence) located in luminar debris and in the epithelium of an oviduct.
2
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S0093-691X(17)30039-0
DOI:
10.1016/j.theriogenology.2017.01.027
Reference:
THE 13982
To appear in:
Theriogenology
Received Date: 1 November 2016 Revised Date:
13 January 2017
Accepted Date: 15 January 2017
Please cite this article as: Karstrup CC, Pedersen HG, Jensen TK, Agerholm JS, Bacterial invasion of the uterus and oviducts in bovine pyometra, Theriogenology (2017), doi: 10.1016/ j.theriogenology.2017.01.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Revised
1
Bacterial invasion of the uterus and oviducts in bovine pyometra
2
C. C. Karstrupa, H. G. Pedersena, T. K. Jensenb, J. S. Agerholma,1.
3 4
a
5 6
b
RI PT
Department of Large Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Dyrlægevej 68, DK – 1870 Frederiksberg, Denmark National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, DK – 1870 Frederiksberg, Denmark
7
Keywords: Domain Bacteria, Fusobacterium necrophorum, Porphyromonas levii, Trueperella pyogenes, Cow, Fluorescence in situ hybridization.
SC
8 9 10
Abstract
12 13 14 15 16 17 18 19 20 21 22 23
Pyometra is a common disease of cattle that causes infertility and thereby financial losses to the cattle industry. Bacteria involved in the development and progression of pyometra have been investigated by microbial culture but their tissue invading abilities, which is an important aspect of bacterial pathogenicity and development of lesions, have not been investigated. Bacterial invasion of the uterus and oviducts was studied in 21 cows diagnosed with pyometra at the time of slaughter by applying fluorescence in situ hybridization using probes targeting 16S ribosomal RNA of Fusobacterium necrophorum, Porphyromonas levii, Trueperella pyogenes and the overall bacterial domain Bacteria. Fusobacterium necrophorum and P. levii were found to invade the endometrium, especially if the endometrium was ulcerated, and penetrated deep into the lamina propria. These species co-localized within the tissue thus indicating a synergism. Trueperella pyogenes did not invade the uterine tissue. In addition to endometrial lesions, most cows with pyometra also had salpingitis but without significant bacterial invasion of the oviductal wall.
TE D
M AN U
11
25 26
AC C
EP
24
27
1
28
E-mail address: [email protected] (J.S. Agerholm)
Corresponding author Tel.: +4529178136
1
ACCEPTED MANUSCRIPT Revised
1. Introduction
30 31 32 33 34 35
Endometrial bacterial contamination develops in up to 90% of cows after parturition [1]. Bacterial contamination can progress into a postpartum infection, leading to conditions such as endometritis, metritis, and pyometra [2]. The diseases are associated with reduced reproductive performance, reduced milk production, milk withdrawal and increased costs for treatment leading to economic losses [3–12]. The annual costs of bovine postpartum uterine infections were estimated to be around €1.4 billion in the EU and around $650 million in the United States in 2009 [12].
36 37 38 39 40 41 42 43 44 45 46
Pyometra accounts for 2% to 5% of the clinical cases of uterine diseases in cows [13,14] and is characterized by inflammation of the endometrium and accumulation of a purulent exudate in the uterine lumen in the presence of a persistent corpus luteum and a closed cervical canal [15,16]. Cows that ovulate early in the postpartum period, when bacterial contamination is still present in the uterus, are at higher risk of developing pyometra [17]. If endometritis is present after ovulation and during diestrus, the inflammation may inhibit the endometrial prostaglandin production thus preventing corpus luteum regression [16,17]. This inhibition causes a permanent closure of the cervical canal and subsequent accumulation of pus in the uterine lumen [16,17]. Affected cows show no systemic signs of illness, but are infertile [18]. Pyometra is usually treated by injection of PGF2α, which induces regression of the corpus luteum leading to relaxation of the cervix and expulsion of the intrauterine exudate with subsequent estrus as a result [19].
SC
M AN U
EP
TE D
The bacterial flora in cases of bovine pyometra has been determined using conventional culture methods [17,20–23] and recently by culture-independent methods [24]. The most frequently isolated bacteria cultured from cases of pyometra are Trueperella pyogenes and Gram-negative bacteria [20,21], especially Fusobacterium necrophorum [17]. In addition to these frequently isolated bacteria, Escherichia coli, Bacteroides melaninogenicus, streptococci, staphylococci, Pasteurella haemolytica, Bacillus spp. and Diphtheroides spp. have been cultured [17,21]. The central role of T. pyogenes has been shown experimentally by inducing pyometra in postpartum cows either by inoculation of a monoculture or together with F. necrophorum and B. melaninogenicus [22,23]. By using next generation sequencing, we found bacteria belonging to the families Fusobacteriaceae (includes F. necrophorum), Mycoplasmataceae, Bacteroidaceae, Porphyromonadaceae and Pasteurellaceae in cases of bovine pyometra with Fusobacteriaceae as the most frequently found bacterium [24]. Actinomycetaceae and Enterobacteriaceae, which include T. pyogenes and E. coli, respectively, were however only a minor part of the operational taxonomic units [24].
AC C
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69
RI PT
29
Culturing and sequencing methods are useful to provide information on bacteria present in cases of pyometra. We have previously applied bacterial sequence analysis to the luminal content and endometrium of cows with pyometra [24], while other studies have focused only on the luminal content [17,20,24] and therefore not provided insight into the invasive properties of the bacteria present. Assessment of invasive properties is important as invasiveness may be associated with development of chronic endometrial lesions influencing the prognosis for fertility. Furthermore, treatment with antibiotics after restoration of cyclicity by PGF2α may be more effective if targeting tissue invading bacteria.
2
ACCEPTED MANUSCRIPT Revised
70 71 72
Therefore, we used fluorescence in situ hybridization (FISH) to study the localization of bacteria in the stroma of the uterus and oviducts in cases of bovine pyometra and to determine to which extent F. necrophorum, T. pyogenes and Porphyromonas levii invade these structures.
RI PT
73
2. Materials and Methods
75 76 77 78 79 80 81 82
2.1 Animals Reproductive tracts of dairy and beef cows with pyometra were collected at a slaughterhouse and sent to the University of Copenhagen for examination from August 2012 to August 2013. Cooling was initiated at the slaughterhouse shortly after slaughter and the organs were kept at 4 ºC during storage and transportation. Inclusion criteria were: 1) the cow was slaughtered more than 21 days postpartum, 2) the uterus was distended with pus, 3) the cervical canal was closed and 4) a corpus luteum was present in one or both ovaries.
83
2.2 Sampling
84 85 86 87 88 89
Before opening the uterus, the serosal surface was wiped with 70% ethanol and flame sterilized at the incision point. A full thickness specimen of about 1x1 cm was taken from the dorsal aspect of the base of the left uterine horn using sterile forceps and a scalpel. The uterine sample and the entire oviducts were fixed in 10% neutral buffered formalin. An endometrial tissue sample (0.5 x 0.5 cm) and 1 mL of luminal exudate were also sampled and used for 16S rRNA polymerase chain reaction combined with next generation sequencing. These results have been published separately [24].
TE D
90
M AN U
SC
74
2.2.1 Histology
92 93 94 95
The formalin fixed specimens were trimmed and from the oviducts, the ampullae were isolated. The tissues were processed by routine methods, embedded in paraffin, sectioned at 3 µm and stained with hematoxylin and eosin. Selected specimens were also stained by Luna’s method for eosinophilic granula.
EP
91
97 98 99 100 101 102
AC C
96
2.2.2 FISH
Sections for FISH were mounted on SuperFrost®Plus slides (Menzel-Gläser, Germany), and 16S rRNA oligonucleotide probes targeting F. necrophorum, P. levii, T. pyogenes and the overall bacterial domain Bacteria (Eub-338) were used (Table 1). Positive and negative controls were included in each hybridization. Positive controls consisted of sections containing the specific bacterial species and processed with the respective probes, while negative controls were not processed with the probe.
103
3
ACCEPTED MANUSCRIPT Revised
The hybridizations were performed in Shandon hybridization racks (Thermo Scientific, UK), using 99 µL in situ hybridization buffer (1 M Tris (pH 7.2), 5 M NaCl, 10% SDS, H2O) added 1 µL probe. The slides were incubated at 46 °C for 16 to 24 h, rinsed 3 x 3 min with in situ hybridization buffer and 3 x 3 min with in situ washing solution (1 M Tris (pH 7.2), 5 M NaCl, H2O), and finally dipped in MilliQ water 10 to 15 times at room temperature.
109 110 111 112 113 114
The slides were mounted with Vectashield® (Vector Laboratories, INC. Burlingame, CA, USA), and evaluated using an Axiomager M1 epifluorescence microscope with a 100-W HBO lamp. Filter sets 43 and 38 were used to visualize Cy3 (excitation/emission peak at 552/570 nm) and FITC (excitation/emission peak at 495/520 nm), respectively. Images were obtained using an AxioCam MRm version 3 FireWiremonocrome camera and AxioVision software, version 4.5 (Carl Zeiss, Oberkochen, Germany).
115 116 117
The slides were evaluated by one examiner (CCK) on the day of hybridization at X 400 magnification. The presence and the specific localization of bacteria within the tissue or on the surface of the mucosa were recorded.
M AN U
SC
RI PT
104 105 106 107 108
118 119
2.3 Data management
120
Calving date, date of slaughter and parity were extracted from the Danish Cattle Data Base.
121
3. Results
123
3.1 Study population
124 125 126 127 128 129
Twenty-one cows were included in the study. Twenty reproductive tracts were examined within 48 h of slaughter whereas one tract was examined 96 h after slaughter. The left oviduct and ovary from one cow was lost during slaughter. The parity ranged from one to six, with a mean parity of 2.9. The cows were on average 290 days after parturition [range 26 to 1093 days]. One cow had been treated for endometritis (76 days before slaughter) and three others for retained fetal membranes (100, 112 and 462 days before slaughter). Details on treatment were not available.
EP
AC C
130
TE D
122
131
3.2 Histology
132
3.2.1 Uterus
133 134 135 136 137 138
Most of the specimens had a single layer of columnar epithelium through which neutrophils migrated. Endometrial polyps (Fig. 1A) were present in 38.1% (8/21) of the specimens, and 47.6% (10/21) had endometrial ulceration characterized by loss of epithelium, deposits of fibrin and massive infiltration with neutrophils and mononuclear cells in the adjacent lamina propria. Ulceration with development of granulation tissue (Fig. 1B) was present in 19.0% (4/21) of the specimens. These ulcerations were characterized by well-vasculized connective tissue hyperplasia (fibrosis, granulation tissue), which was
4
ACCEPTED MANUSCRIPT Revised
covered by fibrin. In cases without a single layer of columnar epithelium, the epithelium was either missing due to extensive ulceration or the epithelium was altered to a pseudostratisfied columnar epithelium.
142 143
The thickness of the lamina propria was generally normal but was atrophic in five cases (Fig. 1C) where endometrial fibrosis, non-suppurative inflammation and atrophy of glands were present.
144 145 146 147 148
The lamina propria was infiltrated by mononuclear cells and neutrophils in most cases (19/21). Infiltration was often diffuse and affected the upper 25% to 50% of the lamina propria, but in four cases the inflammation involved the entire lamina propria. Nodular lymphoid hyperplasia (Fig. 1C) was present in 61.9% (13/21) of the specimens. Two specimens did not have inflammation of the lamina propria at all.
149 150 151 152 153
Endometrial glands were evenly distributed over the entire specimen in 17 cases, while 3 cases had a multifocal distribution and in one case glands were completely absent. The glands were coiled and covered by tall columnar epithelial cells, except for one case where the glandular epithelium was flattened. Periglandular fibrosis (Fig. 1C) was observed in 47.6% (10/21) of the specimens. Tunica muscularis and the serosa were not affected in any of the specimens.
M AN U
SC
RI PT
139 140 141
154
3.2.2 Oviducts
156 157 158 159 160 161
A folded luminal surface with a single layer of intact ciliated epithelium was present in all but one oviduct. Either uni- or bilateral ampullar salpingitis was present in 18 cows (Fig. 1D). The inflammation was characterized by a luminal debris containing bacteria (Fig 2B) and cellular infiltration of the superficial part of the lamina propria, mainly by lymphocytes and eosinophils. Scattered neutrophils were present and occasionally macrophages were observed. Nodular lymphoid hyperplasia was present in 9.8% (4/41) of the oviducts.
162 163 164
One oviduct from one cow differed markedly from the others. This oviduct was distended to a diameter about six times greater than usually observed and no folds were present. The epithelium was ulcerated and glands were present in the tunica muscularis (adenomyosis).
165
AC C
EP
TE D
155
166
3.3 FISH
167 168 169
Initially, the positive and negative controls were examined. For each section the borders were examined for presence of bacteria to exclude contamination of the individual section. Contamination was not observed.
170 171 172 173 174
3.3.1. Uterus By using the general bacterial probe Eub-338, bacteria were found on the endometrial surface, in the luminal debris and in the lamina propria (Fig. 2A). Bacteria were present in the lumen of the uterus in all cows and within the endometrium in 42.9% (9/21) of the cows (Table 2).
5
ACCEPTED MANUSCRIPT Revised
Fusobacterium necrophorum and T. pyogenes were both found in 76.2% (16/21) of the cows. While F. necrophorum was tissue invasive in 56.3% (9/16) of the cases, T. pyogenes was predominantly (87.5%; 14/16) found in the lumen or on the endometrial surface. Porphyromonas levii was less prevalent (33.3%) but when present, this bacterium had invaded the endometrium in all cases (Table 2). Bacterial invasion of the lamina propria was only observed in relation to endometrial ulcers. While T. pyogenes only invaded the outermost part of the lamina propria, F. necrophorum and P. levii penetrated deep into the lamina propria. Examination for co-localization of the bacteria revealed that F. necrophorum and T. pyogenes did not co-localize in the tissue, but P. levii was consistently located close to F. necrophorum.
185
3.3.2 Oviducts
186 187 188 189 190 191 192 193
Bacteria were present in at least one oviduct in 76.2% (16/21) of the cows when the general Eub-338 probe was applied. Of these, seven cows (43.8%) had bacteria in the ampulla unilaterally and nine cows (56.2%) had bilateral infection. The bacteria were primarily located on the epithelial surface and in the luminal debris (Table 3; Fig. 2B). Fusobacterium necrophorum, P. levii and T. pyogenes were found in the outermost part of the lamina propria in only one case with ulceration of the ampulla. Otherwise the bacteria were highly prevalent intraluminally (Table 3).
194
4. Discussion
195 196 197 198 199 200 201 202 203
FISH examination of uterine tissue samples revealed that F. necrophorum and T. pyogenes were present in most cases (76.2%), but their location differed as T. pyogenes was predominantly present in the uterine lumen or on the endometrial surface while F. necrophorum was tissue invasive in approximately half of the cases. Porphyromonas levii was less prevalent (33.3%), but when present this bacterium had invaded the tissue in all cases. Knudsen and coworkers [24], who examined endometrial specimens of the same cows, observed that the highest number of operational taxonomic units belonged to the family Fusobacteriaceae (including F. necrophorum) in accordance with the FISH results of the present study. Also, culture based studies have shown that F. necrophorum is frequently present in cases of bovine pyometra [17,20,21].
205 206 207 208 209 210 211 212
SC
M AN U
TE D
EP
AC C
204
RI PT
175 176 177 178 179 180 181 182 183 184
Porphyromonas levii has not previously been isolated from the uterus of cows with pyometra, but we included this species in the present study as it often co-localized with F. necrophorum in bovine postpartum endometritis (unpublished results) and because bacterial sequencing found a large number of reads from the family Porphyromonadaceae, which includes P. levii [24]. In addition to F. necrophorum and T. pyogenes, a wide range of other bacterial species have also been identified in culture based studies [17,20,21]. Similarly, bacteria other than F. necrophorum, P. levii and T. pyogenes, i.e. bacteria only identified by using the Eub-338 probe, accounted for approximately 2/3 of the overall bacterial load in the uterine specimens in the present study.
6
ACCEPTED MANUSCRIPT Revised
Culture-based studies of bovine pyometra have been performed on the uterine luminal content [17,20,21]. However, in this study, we focused on the tissue invading bacteria. Among the pathogens investigated, only F. necrophorum and P. levii had invaded the endometrium while T. pyogenes was mainly present in the lumen or on the endometrial surface. When comparing the results from applying the general bacterial probe (Eub-338) and the specific probes for F. necrophorum and P. levii, these latter species were found to be the only, or the absolutely dominating, bacterial species penetrating deep into the lamina propria, thus emphasizing their role in causing tissue damage in bovine pyometra. The co-localization of these species indicates a synergism that may exacerbate their pathogenicity. Such synergism has also been reported by others [25,26]. The tissue invading properties may be of importance as the extent of tissue damage likely will affect the cow’s ability to become pregnant when reproductive cyclicity is restored after treatment with PGF2α. Treatment with a combination of PGF2α and either oxytetracycline or cephapirin has been shown to increase pregnancy rates compared to nontreated cows [27]. Trueperella pyogenes was not found within the endometrium and thus did not show significant tissue invading properties, but the bacterium may play a role in the development and progression of pyometra through production of exotoxins such as pyolysin [28,29]. The invasive properties and co-localization of F. necrophorum and P. levii and the luminal localization of T. pyogenes found in this study are similar to findings in a study of bovine postpartum endometritis (unpublished results).
231 232 233 234 235 236 237
The presence of bacteria and inflammation in oviducts from cows with pyometra has not been reported previously, although damage to the oviducts is likely to influence the subsequent fertility even if cyclicity is regained. We found uni- or bilateral bacterial infection or salpingitis as detected by histology in 76.2% and 69.2% of the examined cows, respectively. Bacterial infection of the oviducts and salpingitis may therefore be common in bovine pyometra. However, lesions were generally less severe in the oviducts than in the uterus and restoration of a normal function may be possible if a normal uterine function and environment is achieved.
238
TE D
M AN U
SC
RI PT
213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230
5. Conclusions
240 241 242 243 244
In the present study we showed that F. necrophorum and P. levii penetrate deeply into the endometrium in bovine pyometra, especially if the endometrium is ulcerated. Also, these bacterial species seem to display a synergism as they co-localize in the tissue. Trueperella pyogenes does not display significant tissue invading properties but may participate in the development and progression of pyometra by other mechanisms. In addition to endometrial lesions, most cows with pyometra develop salpingitis.
AC C
245
EP
239
246
Acknowledgement
247 248 249
We thank the staff at DC Tønder for participating in this study by submitting materials of slaughtered cows. The technical assistance of Mrs. Annie Ravn Pedersen, Mrs. Heidi Holm and Mrs. Anne Dorte Roed is greatly acknowledged.
250
7
ACCEPTED MANUSCRIPT Revised
251
Funding
252 253
This study was performed as part of a PhD program funded by the University of Copenhagen and financially supported by Mælkeafgiftsfonden.
255
Competing interests
256
The authors declare that they have no competing interests.
257
RI PT
254
References
259 260 261
[1]
Griffin JFT, Hartigan PJ, Nunn WR. Non-specific uterine infection and bovine fertility I. Infection patterns and endometritis during the first seven weeks post-partum. Theriogenology 1974;1:91–106.
262
[2]
Sheldon IM. Post-partum uterine infections and their treatment. Cattle Pract 1999;7:23–4.
263 264 265
[3]
Salasel B, Mokhtari A, Taktaz T. Prevalence, risk factors for and impact of subclinical endometritis in repeat breeder dairy cows. Theriogenology 2010;74:1271–8. doi:10.1016/j.theriogenology.2010.05.033.
266 267 268
[4]
Leblanc SJ, Duffield TF, Leslie KE, Bateman KG, Keefe GP, Walton JS, et al. Defining and diagnosing postpartum clinical endometritis and its impact on reproductive performance in dairy cows. J Dairy Sci 2002;85:2223–36. doi:10.3168/jds.S0022-0302(02)74302-6.
269 270 271
[5]
Williams EJ, Fischer DP, Noakes DE, England GCW, Rycroft A, Dobson H, et al. The relationship between uterine pathogen growth density and ovarian function in the postpartum dairy cow. Theriogenology 2007;68:549–59. doi:10.1016/j.theriogenology.2007.04.056.
272 273
[6]
Sheldon M, Noakes DE, Rycroft AN, Dobson H, Sheldon IM. Effect of postpartum manual examination of the vagina on uterine bacterial contamination in cows. Vet Rec 2002;151:531–4.
274 275
[7]
Barlund CS, Carruthers TD, Waldner CL, Palmer CW. A comparison of diagnostic techniques for postpartum endometritis in dairy cows. Theriogenology 2008;69:714–23.
276 277
[8]
Hammon DS, Holyoak GR, Jenson J, Bingham HR. Effects of endometritis at the begining of the breeding period on reproductive performance in dairy cows. AABP Proc 2001;34:142–3.
278 279 280
[9]
Studer E, Morrow Da. Postpartum evaluation of bovine reproductive potential: comparison of findings from genital tract examination per rectum, uterine culture, and endometrial biopsy. J Am Vet Med Assoc 1978;15172:489–94.
281 282 283
[10]
Gilbert RO, Shin ST, Guard CL, Erb HN, Frajblat M. Prevalence of endometritis and its effects on reproductive performance of dairy cows. Theriogenology 2005;64:1879–88. doi:10.1016/j.theriogenology.2005.04.022.
284 285
[11]
Borsberry S, Dobson H. Periparturient diseases and their effect on reproductive performance in five dairy herds. Vet Rec 1989;124:217–9.
286
[12]
Sheldon IM, Cronin J, Goetze L, Donofrio G, Schuberth H-J. Defining postpartum uterine
AC C
EP
TE D
M AN U
SC
258
8
ACCEPTED MANUSCRIPT Revised
disease and the mechanisms of infection and immunity in the female reproductive tract in cattle. Biol Reprod 2009;81:1025–32. doi:10.1095/biolreprod.109.077370.
287 288
[13]
Mickelsen WD, Paisley LG, Anderson PB. Survey of the prevalence and types of infertility in beef cows and heifers. J Am Vet Med Assoc 1986;1:51–4.
291 292
[14]
Sheldon IM, Williams EJ, Miller ANA, Nash DM, Herath S. Uterine diseases in cattle after parturition. Vet J 2008;176:115–21. doi:10.1016/j.tvjl.2007.12.031.
293 294
[15]
Sheldon IM, Lewis GS, LeBlanc S, Gilbert RO. Defining postpartum uterine disease in cattle. Theriogenology 2006;65:1516–30. doi:10.1016/j.theriogenology.2005.08.021.
295 296
[16]
Bondurant RH. Inflammation in the bovine female reproductive tract. J Anim Sci 1999;77:101– 10.
297 298 299
[17]
Olson JD, Ball L, Mortimera RG, Farin PW, Adney WS, Huffman EM. Aspects of bacteriology and endocrinology of cows with pyometra and retained fetal membranes. Am J Vet Res 1984;45:2251–5.
300 301
[18]
Palmer C. Postpartum uterine infection. Bov. Reprod., 2014, p. 440–8. doi:10.1002/9781118833971.ch50.
302 303
[19]
Gustafsson BK, Bäckström G, Edqvist LE. Treatment of bovine pyometra with prostaglandins: An evaluation of a field study. Theriogenology 1976;6:45–50.
304 305
[20]
Noakes DE, Wallace LM, Smith GR. Pyometra in a Friesian heifer: Bacteriological and endometrial changes. Vet Rec 1990;126:509.
306 307 308
[21]
Brodzki P, Kostro K, Brodzki A, Niemczuk K, Lisiecka U. Cytometric analysis of surface molecules of leucocytes and phagocytic activity of granulocytes and monocytes/macrophages in cows with pyometra. Reprod Domest Anim 2014;49:858–64. doi:10.1111/rda.12381.
309 310 311
[22]
Davidson J a, Wright DJ, Archbald LF, Klapstein E, Gottshall SL, Hansen PJ. Effect of induced pyometra on luteal lifespan and uterine fluid concentrations of prostaglandins and interferons in cows. Theriogenology 1996;45:459–70. doi:10.1016/0093-691X(95)00382-I.
312 313 314
[23]
Farin PW, Ball L, Olson JD, Mortimer RG, Jones RL, Adney WS, et al. Effect of Actinomyces pyogenes and Gram-negative anaerobic bacteria on the development of bovine pyometra. Theriogenology 1989;31:979–89. doi:10.1016/0093-691X(89)90481-0.
315 316 317
[24]
Knudsen LRV, Karstrup CC, Pedersen HG, Agerholm JS, Jensen TK, Klitgaard K. Revisiting bovine pyometra - New insights into the disease using a culture-independent deep sequencing approach. Vet Microbiol 2015;175:319–24. doi:10.1016/j.vetmic.2014.12.006.
318 319
[25]
Kan IY, Blum S, Elad D. Synergism between Porphyromonas levii and Arcanobacterium pyogenes in a murine abscess model. Isr J Vet Med 2009;64: En62-En65, He23-24.
320 321
[26]
Bekana M, Jonsson P, Ekman T, Kindahl H. Intrauterine bacterial findings in postpartum cows with retained fetal membranes. J Vet Med Ser A 1994;41:663–70.
322 323 324
[27]
El-Tahawy AEGS, Fahmy MM. Partial budgeting assessment of the treatment of pyometra, follicular cysts and ovarian inactivity causing postpartum anoestrus in dairy cattle. Res Vet Sci 2011;90:44–50. doi:10.1016/j.rvsc.2010.04.017.
325
[28]
Jost BH, Billington SJ. Arcanobacterium pyogenes: molecular pathogenesis of an animal
AC C
EP
TE D
M AN U
SC
RI PT
289 290
9
ACCEPTED MANUSCRIPT Revised
opportunist. Int J Gen Mol Microbiol 2005;88:87–102. doi:10.1007/s10482-005-2316-5.
326 327 328 329
[29]
Amos MR, Healey GD, Goldstone RJ, Mahan SM, Düvel A, Schuberth H-J, et al. Differential endometrial cell sensitivity to a cholesterol-dependent cytolysin links Trueperella pyogenes to uterine disease in cattle. Biol Reprod 2014;90:54. doi:10.1095/biolreprod.113.115972.
RI PT
330
AC C
EP
TE D
M AN U
SC
331
10
ACCEPTED MANUSCRIPT Revised
1
Tables
2
4 5
Table 1: Oligonucleotide probes* used for locating bacteria in uterine and oviductal tissue in cows with pyometra. Name of the probe
Target sequence (5´-3´)
Domain Bacteria Fusobacterium necrophorum Porphyromonas levii Trueperella pyogenes
S-D-Eub-338 S-S-F.necrop-183 P.levii-443 S-S-A.pyogenes-a-A
5´ gctgcctcccgtaggagt 3´ 5´ gattcctccatgcgaaaa 3´ 5´ tacctacgtttactcgcc 3´ 5´ gcacataccgtcacaaaa 3´
M AN U
*Eurofins MWG Operon, Ebersberg, Germany.
7 8 9
Table 2: Location of bacteria in the uterus of cows with pyometra (n = 21) determined by fluorescence in situ hybridization (FISH) using probes for Fusobacterium necrophorum, Porphyromonas levii, Trueperella pyogenes and the overall bacterial domain Bacteria.
TE D
10 11 12
Target Fluorochromes References region 16s FITC [25,26] 16s FITC [26] 16s Cy3 [27] 16s FITC
SC
Target bacteria
6
RI PT
3
14
AC C
13
Localization of bacteria Both luminally* Only Only in the lamina and in the lamina luminally* propria propria Domain Bacteria 100.0% (21/21) 42.9% (9/21) 57.1% (12/21) 0% F. necrophorum 76.2% (16/21) 56.3% (9/16) 43.8% (7/16) 0% P. levii 33.3% (7/21) 100.0% (7/7) 0.0% (0/7) 0% T. pyogenes 76.2% (16/21) 12.5% (2/16) 87.5% (14/16) 0% *either in luminal debris or on the endometrial surface
EP
FISH positive cows
1
ACCEPTED MANUSCRIPT Revised
15
Table 3: Location of bacteria in the oviductal ampulla of cows with pyometra (n = 21) determined by fluorescence in situ hybridization (FISH) using probes for Fusobacterium necrophorum, Porphyromonas levii, Trueperella pyogenes and the overall bacterial domain Bacteria.
RI PT
16 17 18
FISH positive cows (Uni- or bilaterally)
20
SC
Only in the lamina propria 0% 0% 0% 0%
Figure legends
21 22
EP
TE D
Figure 1. Photomicrographs of lesions in the endometrium and oviducts of cows with pyometra. A. An endometrial polyp (P) protrudes into the uterine lumen. The polyp is covered by a pseudostratisfied columnar epithelium and the lamina propria is heavily infiltrated with mononuclear inflammatory cells (asterisk). B. Chronic endometritis characterized by presence of inflamed granulation tissue (G) covered by fibrin (arrowheads) and infiltration with neutrophils (arrows). C. Chronic endometritis characterized by diffuse endometrial atrophy, fibrosis (F) and focal accumulation of lymphocytes (asterisk). Glands are absent except for a single gland with periglandular fibrosis (arrow). D. Suppurative inflammation of the oviduct. Numerous neutrophils migrate through the epithelium (arrowheads) and accumulate in the lumen (arrows). A-D: Hematoxylin and eosin.
AC C
23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Both luminally* Only luminally* and in the lamina propria 12.5% (2/16) 87.5% (14/16) 8.3% (1/12) 91.7% (11/12) 14.3% (1/7) 85.7% (6/7) 6.3% (1/16) 93.7% (15/16)
M AN U
19
Domain Bacteria 76.2% (16/21) F. necrophorum 81.3% (13/16) P. levii 100.0% (7/7) T. pyogenes 68.8% (11/16) *either in luminal debris or on the mucosa
Localization of bacteria
Figure 2. Fluorescence in situ hybridization photomicrographs
A. Endometrial ulceration with numerous bacteria (green fluorescence) invading the tissue. General bacteria probe (Domain Bacteria, Eub-338). Yellow fluorescence represents autofluorescence omitted by erythrocytes. B. Fusobacterium necrophorum (green fluorescence) and Porphyromonas levii (red fluorescence) located in the lumen of the oviduct. C. Trueperella pyogenes (red fluorescence) and other bacteria (Domain Bacteria, Eub-338; green fluorescence) located in luminar debris and in the epithelium of an oviduct.
2
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT