2009: Endometritis in dogs - current knowledge and future considerations Endometritis in dogs – current knowledge and future considerations 1 S.K. Lyle 2 Department of Veterinary Clinical Sciences School of Veterinary Medicine, 3 Louisiana State University, Baton Rouge, LA 4 Abstract 5 Canine endometritis, as an entity separate from cystic endometrial hyperplasia, is 6 a poorly understood process, yet may be responsible for infertility. Inflammatory 7 infiltrates of the endometrium in the absence of proliferative changes are the hallmark of 8 this disease. The ascension of bacteria from the vagina through the cervix is theorized 9 to lead to a chronic, low-grade endometritis, which does not produce overt clinical 10 disease, such as that seen with pyometra. Escherichia coli is the organism most 11 commonly implicated in pyometra, and is likely the most common organism involved 12 with endometritis. Various uropathogenic virulence factors (UVFs) have been identified 13 from uterine isolates, many of which share common UVFs from isolates producing 14 urinary tract disease in dogs and humans. Of these, P fimbria is thought to be crucial for 15 initial bacterial adherence to the luminal epithelium. Reduction in Mucin-1 expression 16 and immunolocalization in endometrial epithelial cells in early diestrus may be involved 17 with producing a permissive state for bacterial adhesion, allowing colonization. Samples 18 that may aid in the diagnosis of endometritis include vaginal or uterine cultures, uterine 19 biopsy, and possibly endometrial cytology and ultrasonography. Treatment of 20 endometritis is focused on elimination of infection through the use of antimicrobial 21 agents, and physical clearance of the uterus through the use of prostaglandins, 22 dopamine agonists, and progesterone-receptor antagonists. 23 105 24 Keywords: Dog, infertility, endometritis, diagnosis, treatment 25 Introduction 26 Although many clinicians might empirically agree that inflammation of the 27 endometrium may contribute to a reduction in fertility, direct evidence of a causal effect 28 of endometritis on reducing fertility is lacking. Subfertility and infertility due to cystic 29 endometrial hyperplasia is more generally agreed upon; however, whether the reduction 30 in fertility is due to the proliferative response of the endometrium or the inflammatory 31 component of the process is unknown. 32 Histopathology of the canine endometrium 33 Endometrial hyperplasia, with or without cystic changes is considered a post-34 estrual luteal phase disease. Age and nulliparity are considered risk factors for 35 development of CEH.1,2 Two main hypotheses exist on the pathogenesis of CEH and 36 subsequent development of pyometra. The classical description involves the 37 development of endometrial hyperplasia which subsequently leads to an inflammatory 38 reaction. The accumulation of secretions from the hyperplastic endometrium supports 39 the growth and proliferation of bacteria which ascend through the cervix. Establishment 40 of bacterial infection leads to the accumulation of exudate (pyometra). An alternative 41 hypothesis centers on the premise that chronic low-grade uterine infection drives the 42 endometrial proliferative response either by bacterial toxins or inflammatory mediators.3 43 It has also been suggested that endometritis-pyometra can occur independent of 44 endometrial hyperplasia; unfortunately the age distribution of these cases were not 45 defined.4 A variety of stimuli have been used to experimentally reproduce endometrial 46 106 proliferation. These have ranged from china balls,5 suture,6-8 and bacteria,9-13 among 47 others. The proliferative changes observed have been well-characterized, and are not 48 the focus of this discussion. For detailed information on CEH, the reader is directed to 49 several excellent reviews on the topic.1,3,4,14 50 Endometritis is also a common finding, and was recently determined to be the 51 most common diagnosis (94 cases) in a survey of 366 canine endometrial biopsies. 52 Hyperplasia was the second most common diagnosis (86 cases).15 It is most common 53 to find a plasmacytic infiltrate with subclinical endometritis14 or with CEH with 54 plasmacytic infiltration and little intraluminal fluid accumulation,1 progressing to 55 neutrophilic plasmacytic infiltrates in cases of CEH and significant fluid accumulation 56 (pyometra).1 57 Microflora of the reproductive tract 58 Debate exists on whether vaginal cultures are useful for determining the 59 presence of intrauterine infection. Although most prepuberal and postpuberal bitches 60 were found to have positive vaginal cultures, the majority of uterine cultures were found 61 to be negative.16 Similarly, all uterine swabs collected from late diestrus, progestin-62 supplemented bitches were sterile,17 although concurrent sampling of the vagina was 63 not performed. The predominate isolate from infected uteri is Escherichia coli,1,18-22 with 64 reported incidences of 73%,1 79.4%,21 and 85%.22 Although E. coli in pure culture was 65 the most common isolate from bitches with pyometra, mixed cultures were the most 66 common finding in ‘infertile’ bitches, leading to the conclusion that vaginal cultures have 67 low diagnostic value.20 Conversely, other studies found the uterus not to be a sterile 68 environment,23,24 and that vaginal isolates reflected those of the uterus.23 Bacteria were 69 107 consistently recovered from the uterus during proestrus and estrus, and post-mortem 70 uterine isolates always reflected those of the cervix and vagina.24 The most common 71 uterine isolates were E. coli, Haemophilus spp., α-hemolytic streptococci, 72 Corynebacterium spp., Streptococcus canis, Alcaligenes faecalis, Bacteroides spp., 73 Pasteurella spp., and Proteus mirabilis.24 A method to transcervically collect uterine 74 secretions that is guarded from vaginal secretions would enhance the ability of clinicians 75 to accurately diagnose the presence of bacteria in the uterus. 76 Several authors have investigated virulence factors of E. coli isolates from cases 77 of pyometra. Early characterizations focused on the presence of the O-18 and K-78 antigen21,22 Uropathogenic E. coli strains, which are responsible for urinary tract 79 infections in dogs and cats, may originate from the intestinal tract, and possess a cluster 80 of virulence-related genes encoding for specific O-antigens, type 1 fimbriae, P fimbriae, 81 S fimbriae, α-hemolysin, cytotoxic necrotizing factor 1, and aerobactin (iron-82 sequestering system).25-27 These strains are not canine-specific, and it has been 83 suggested that the dog may serve as a source of uropathogenic E. coli for human 84 urinary tract infections (UTI).28,29 Biochemical fingerprinting of E. coli isolates from 85 pyometra and UTI suggest that these isolates originate from the fecal flora, and the 86 same clone of E. coli is present in cases with concurrent UTI and pyometra.30 87 Similarly, DNA-profiles of E. coli isolates from the urinary bladder and uterus of bitches 88 affected simultaneously with UTI and pyometra were 100% identical, and that all 89 colonies from a site were identical, despite macroscopic morphologic differences.31 The 90 papGIII allele, the most frequent allele encoding for P fimbriae in canine and human 91 uropathogenic E. coli isolates,29 had a significantly higher prevalence in E. coli isolates 92 108 from pyometra, and the proportion of strains from pyometra possessing more than three 93 uropathogenic factors was greater than that of fecal strains.32 The presence of P 94 fimbriae is thought to be crucial for bacterial adherence to epithelial cells of the urinary 95 tract. The PapGIII adhesion binds to Galα1-4 Galβ-containing glycolipid receptor and its 96 coreceptor TLR4 present on urinary epithelial cells.33 The presence of these receptors 97 has been confirmed for canine urinary epithelium,34 but has not been investigated in 98 canine endometrium. Other virulence genes that have been associated with 99 uropathogenicity and were present in high proportions of isolates from pyometra 100 isolates include fim (Type I fimbriae) and sfa (S fimbriae), although the differences were 101 not statistically significant.32 Although Type I fimbriae are present on many isolates 102 from human UTIs, the correlation with pathogenicity is considered low.35 Escherichia 103 coli bearing S fimbriae bind to human renal proximal tubular cells,36 but only 27.4% of 104 strains were positive for sfa compared to 97.5% of strains carrying fimH (Type I 105 fimbriae).37 106 These findings have led researchers to the conclusion that the pathogenesis of 107 both UTI and pyometra involve ascension of intestinal strains of E. coli into the lower 108 urinary tract, cranial vagina, and uterus.28,30,32 Only a few investigators have attempted 109 to induce infection by inoculation with E. coli. In a series of investigations using an E. 110 coli isolate from a clinical case of pyometra, Nomura et al. inoculated the uterus of dogs 111 in either pro-estrus/estrus, diestrus, post-partum, or anestrus with or without cervical 112 ligation.9,11,12 When examined 12 d post-inoculation with cervical ligation, the incidence 113 of pyometra in proestrus/estrus, diestrus, post-partum, and anestrus was 100%, 100%, 114 80%, and 28%, respectively.9 When examined 12 d post-inoculation without cervical 115 109 ligation, the incidence of pyometra in pro-estrus/estrus, diestrus, post-partum, and 116 anestrus was 25%, 89.9%, 70.6%, and 50.6%, respectively. More recently, 5×107 CFU 117 of an E. coli (O2:H:K) isolated from a clinical case of pyometra was inoculated in the 118 uterus of intact bitches at either post-LH day 1-10, 11-20, 21,30, 31-40, 41-50, or 51-60; 119 the incidence of pyometra induced was 16.7%, 90.9%, 78.9%, 62.5%, 40.0%, and 0%, 120 respectively. Bitches with induced pyometra were either treated with dinoprost 121 tromethamine and enrofloxacin, or were allowed to spontaneously recover. There was 122 no difference in pregnancy rates between treated and non-treated bitches on the 123 subsequent estrus, and recurrence of pyometra did not occur.13 While this model did 124 induce pyometra in the strict sense of the definition (the presence of pus in the uterus 125 during the luteal phase), the subsequent fertility and lack of recurrence do not fit the 126 typical clinical scenario of bitches with spontaneous pyometra. This model appears to 127 more closely approximate endometritis than pyometra. Subsequently, inoculation of the 128 uterus with an E. coli strain possessing five UVFs induced CEH/pyometra in diestrus-129 simulated ovariectomized bitches, while inoculation into the vagina failed to establish 130 uterine infection or endometrial changes.10 Differences in these two studies lie in the 131 status of the bitch and potential difference in the pathogenicity of the E. coli strain. 132 Host-pathogen interactions 133 Limited investigation on the host-response to intrauterine infections exists, but a 134 few noteworthy studies shed some light on mechanisms by which bacterial are able to 135 colonize the endometrium. The proliferative response of peripheral blood monocytes 136 (PBMCs) to a clone of E. coli isolated from the uterus from a dog with pyometra was 137 significantly decreased at day 10 of diestrus compared to proestrus, estrus, day 30 of 138 110 diestrus, or anestrus.38 Similarly, the addition of progesterone or 5α-139 dihydroprogesterone to PBMCs collected from anestrous bitches significantly reduced 140 the response to E. coli compared to PBMCs supplemented with estradiol 17-β, 17α-141 hydroxyprogesterone, or pregnenolone; and progesterone reduced the expression of 142 IFNγ by PBMCs compared to estradiol.38 143 Recently lactoferrin, an antimicrobial and immunomodulator member of the 144 transferrin gene family which is expressed by epithelial cells and neutrophil granules, 145 has been identified in the equine and canine endometrium.39,40 Lactoferrin’s 146 antibacterial property lies within its ability to sequester free iron, thereby inhibiting 147 bacterial growth. In the mare, lactoferrin expression was upregulated during early 148 estrus, protein staining was uninfluenced by cycle and was most intense in the 149 glandular epithelium, and expression of lactoferrin was only increased in mares with 150 delayed physical clearance during early estrus,39 which might represent a response to 151 inflammation. The pattern of lactoferrin expression has also been described in the bitch, 152 where expression increased from proestrus to estrus, then significantly decreased from 153 estrus to day 10 of diestrus, remaining low at day 35 of diestrus and anestrus; a similar 154 pattern was observed with immunohistochemical staining for lactoferrin,40 leading the 155 investigators to conclude that estrogen was involved with the regulation of lactoferrin 156 expression. Although reduced lactoferrin expression during diestrus would be a 157 plausible explanation of reduced microbial defenses and increased susceptibility to 158 infection, lactoferrin expression was increased in bitches with pyometra,40 similar to 159 what was observed in mares with delayed uterine clearance and post-mating induced 160 endometritis.39 Increased lactoferrin expression in both instances may due to an influx 161 111 of neutrophils.41 While intriguing from a perspective of host-pathogen interactions, 162 unless diminished response in lactoferrin expression and production were observed in 163 bitches suffering from pyometra, reduced lactoferrin activity is unlikely to be responsible 164 for increased susceptibility to infection. 165 Mucin-1 (Muc1) is an important component of the epithelial cell glycocalyx, 166 functioning as an anti-adhesive molecule; loss of Muc1 expression is considered an 167 integral step in allowing adhesion between the trophoblast and the luminal epithelium.42 168 In normal, cyclic bitches, Muc1 expression and localization was significantly decreased 169 at day 10 of diestrus and in bitches with pyometra compared to proestrus, estrus, day 170 35, or anestrus. Additionally, Muc1 expression and adherence of E. coli to endometrium 171 was inversely correlated.43 Clearly, further research is needed in the area of host-172 pathogen interactions of the canine uterus. 173 Diagnosis of endometritis 174 If bacteria such as E. coli can serve as a stimulus for endometrial proliferation, 175 and hence cystic endometrial hyperplasia; then early diagnosis and appropriate therapy 176 might lead to prolongation of the fertile lifespan of some bitches. For additional detail on 177 specifics for collecting reproductive tract tissues, the reader is directed to an excellent 178 previous review on this topic.44 179 Cultures – The most common method used clinically is guarded culture of the anterior 180 vagina during proestrus. As discussed previously, these may or may not reflect a 181 potential uterine pathogen, but in cases in which significant intrauterine infection is 182 suspected, it is the opinion of this author and others,44 that this method should yield a 183 satisfactory sample. It is also possible to collect intrauterine secretions following 184 112 transcervical catheterization with a 4 to 7 Fr catheter through a rigid cystoscope or 185 endoscope in the standing bitch.45 Unfortunately this sample would also suffer from 186 contamination by vaginal fluids. Hysteroscopy in the anesthetized bitch may lessen the 187 contamination, but caused petechia or ecchymoses in 50% of cases, and poor 188 visualization in 37.5% of cases.45 The method providing the most accurate sample of 189 uterine secretions would be that obtained during hysterotomy. In most instances this 190 sample would be obtained concurrent to uterine biopsy.46 191 Cytology – Endometrial cytology is a commonly used diagnostic tool for the diagnosis of 192 equine endometritis; however, it is not routinely used for evaluation of canine cases. 193 Watts et al.47 described the endometrial cytology of the normal bitch in samples 194 collected by transcervical catheterization or at post-mortem.45 Endometrial cells were 195 present at all stages, and exhibited degenerative changes during late diestrus, anestrus, 196 and postpartum. During proestrus and estrus, healthy endometrial cells, neutrophils and 197 bacteria were commonly observed. During diestrus and early pregnancy, healthy 198 endometrial cells and neutrophils were most common. During late diestrus and 199 anestrus, evidence of endometrial cell degeneration was observed and lymphocytes 200 and macrophages were the most common leukocyte present. To the authors knowledge 201 there are no published reports regarding changes in endometrial cytology with uterine 202 pathology such as endometritis or cystic endometrial hyperplasia. 203 Uterine biopsy – Biopsy of the endometrium is maximally invasive, yet provides the 204 most accurate sample for the diagnosis and prognosis for endometritis or other uterine 205 pathology. Samples can be obtained by either laparotomy,46 or by a transcervical 206 113 approach;48 however, the later technique only provided a diagnostic sample 31% of the 207 time and was associated with hematomucometra in 44% of cases. 208 Ultrasonography – Ultrasonography is commonly used to diagnose cystic endometrial 209 hyperplasia with or without pyometra. Its usefulness for the diagnosis of endometritis 210 has not been correlated with other diagnostic techniques. 211 Treatment of endometritis 212 From the preceding discussion, it is apparent that arriving at an accurate 213 diagnosis of endometritis may be difficult. No controlled studies have been done on 214 therapeutic regimes for the treatment of endometritis. Therefore, treatment options are 215 based on what has been recommended for medical management of pyometra, which is 216 focused on eliminating bacterial infection, if present, and stimulating physical clearance 217 of the uterus. The former is achieved by appropriate antimicrobial agents, the latter by 218 terminating the luteal phase through the use of prostaglandins, dopamine agonists, or 219 progesterone-receptor antagonists, and by stimulation of myometrial contractions 220 through the use of prostaglandins. Prior to initiating medical therapy, detailed owner 221 counseling regarding the intended use and breeding value of the bitch should occur. 222 Ovariohysterectomy should be recommended for bitches without significant 223 reproductive value. 224 Antimicrobial agents – When at all possible, antimicrobial agents should be chosen 225 based on results of culture identification and sensitivity patterns. The injudicious use of 226 antibiotics has the potential to select for strains of bacteria with greater antibiotic 227 resistance patterns. To the author’s knowledge multi-resistant Staphylococcus aureus 228 (MRSA) has not been isolated from a canine case of endometritis or pyometra. This 229 114 would be a dire situation indeed. Recently, MRSA has been recovered from the uterus 230 of mares with extensive history of intrauterine antibiotic therapy.49 Systemic 231 administration in the bitch is the most commonly used route of delivery. Although 232 transcervical delivery of antibiotics into the lumen is possible, it is doubtful that uniform 233 distribution of drug to the entire endometrial surface would occur. Additionally, daily 234 treatment would be required; repeated catheterization adding considerably to the cost of 235 treatment. Systemic administration allows for longer treatment regimes (10-14 d), with 236 good penetration of the endometrium. 237 Prostaglandin – Prostaglandin has been used for many years for the treatment of 238 pyometra.50,51 Typical doses of PGF2α (dinoprost tromethamine) range from 100 to 500 239 µg/kg, SQ, one to three times daily, and are considered luteolytic. Lower doses (10 to 240 50 µg/kg, up to three to five times daily) can also be used to achieve uterine 241 evacuation.52 The use of prostaglandin in the dog is extra-label; therefore, informed 242 client consent is recommended. Transient (20 to 30 min) side effects of salivation, 243 defecation, urination, and emesis are not uncommon at the higher doses, but are 244 drastically reduced when lower doses are used. 245 Dopamine agonists – Inhibitors of prolactin will aid in the rapid reduction in 246 progesterone concentrations, and are frequently combined with low-dose prostaglandin 247 protocols for the treatment of induced abortion.53,54 Bromocriptine (25 µg/kg, PO, q8 to 248 12 h) or cabergoline (5 µg/kg, PO, q24 h), in combination with prostaglandins, can lead 249 to luteolysis with 24 h. 250 Progesterone-receptor antagonists – Although not available in the United States, 251 aglepristone has been used for pregnancy termination,55 and for treatment of pyometra 252 115 alone56 or combination with cloprostenol for pyometra.57 In a recent study the overall 253 success rate for treatment of pyometra (35 open, 17 closed) with cloprostenol (1 µg/kg) 254 and agelpristone (10 mg/kg, daily) was 84.4% compared to 60% for aglepristone alone; 255 the recurrence rate at 12 and 24 mo was 13% and 19% respectively.58 256 Discussion 257 It would seem that there is rather compelling evidence to suggest that subclinical 258 endometritis may precede the development of clinically evident CEH and pyometra. The 259 difficulty lies in the ability to render a diagnosis prior to development of proliferative 260 changes in the endometrium. Clearly further research is needed to prove a causal 261 relationship between these two entities. Comparison of E. coli strains possessing or 262 lacking UVFs in a model would be a useful first step in that process. Further 263 investigation of host-defense mechanisms, such as the presence of toll-like receptors 264 on the endometrium, cytokine signaling involved with endometritis and proliferative 265 changes of the endometrium would also be informative. Biofilms have been described in 266 a variety of mucosal systems, and provide a mechanism for pathogen evasion of host 267 recognition and protection from certain treatment modalities. Uropathogenic E. coli 268 producing biofilms are implicated in chronic urinary tract infections in people,59 and it 269 has been suggested that some cases of E. coli endometritis produce biofilm.60 From a 270 diagnostic standpoint, a guarded system for transcervical collection of uterine secretions 271 in the standing bitch would benefit not only research endeavors, but collection of 272 diagnostic samples from clinical cases. Such information is needed to render an 273 accurate diagnosis, and to progress towards effective treatment strategies. 274 References 275 116 1. Dow C: The cystic hyperplasia-pyometra complex in the bitch. Vet Rec 276 1958;70:1102-1108. 277 2. Egenvall A, Hagman R, Bonnett BN, et al: Breed risk of pyometra in insured dogs 278 in Sweden. J Vet Intern Med 2001;15:530-538. 279 3. Noakes DE, Dhaliwal GK, England GC: Cystic endometrial hyperplasia/pyometra 280 in dogs: a review of the causes and pathogenesis. J Reprod Fertil 281 2001;57(Suppl):395-406. 282 4. De Bosschere H, Ducatelle R, Vermeirsch H, et al: Cystic endometrial 283 hyperplasia-pyometra complex in the bitch: should the two entities be 284 disconnected? Theriogenology 2001;55:1509-1519. 285 5. Krainz K: Über Reizwirkungen von Fremdkörpern auf die Uterusschleimhaut der 286 Hündin. Archiv für Mikroskopische Anatomie 1914;84:122-141. 287 6. Nomura K: Induction of a deciduoma in the dog. J Vet Med Sci 1994;56:365-369. 288 7. Nomura K: Histological evaluation of canine deciduoma induced by silk suture. J 289 Vet Med Sci 1995;57:9-16. 290 8. Chen YM, Wright PJ, Lee CS: A model for the study of cystic endometrial 291 hyperplasia in bitches. J Reprod Fertil 2001;57(Suppl):407-414. 292 9. Nomura K, Kamata Y, Shimada Y: Experimental production of canine pyometra 293 by inoculation of Escherichia coli into the uterus with the cervis ligated: effects of 294 removal of cervical ligature. J Japan Vet Med Assoc 1987;40:842-845. 295 10. Arora N, Sandford J, Browning GF, et al: A model for cystic endometrial 296 hyperplasia/pyometra complex in the bitch. Theriogenology 2006;66:1530-1536. 297 117 11. Nomura K, Funahashi H, Shimada Y: Experimental production of canine 298 pyometra by inoculation of Escherichia coli into the uterus without ligation of the 299 cervix. J Japan Vet Med Assoc 1988;41:95-99. 300 12. Nomura K, Kawata Y, Shimada Y: Experimental production of canine pyometra 301 by inoculation with Escherichia coli into the uterus without ligating the cervix. J 302 Japan Vet Med Assoc 1988;41:17-21. 303 13. Tsumagari S, Ishinazaka T, Kamata H, et al: Induction of canine pyometra by 304 inoculation of Escherichia coli into the uterus and its relationship to reproductive 305 features. Anim Reprod Sci 2005;87:301-308. 306 14. Schlafer DH, Gifford AT: Cystic endometrial hyperplasia, pseudo-placentational 307 endometrial hyperplasia, and other cystic conditions of the canine and feline 308 uterus. Theriogenology 2008;70:349-358. 309 15. Gifford AT, Schlafer DH: Canine uterine biopsy: Endometrial pathology in a 310 population of subfertile bitches, American College of Veterinay Pathologists 311 Annual Meeting. Savannah, GA, 2007. 312 16. Olson PN, Mather EC: Canine vaginal and uterine bacterial flora. J Am Vet Med 313 Assoc 1978;172:708-711. 314 17. Dhaliwal GK, England GC, Noakes DE: The influence of exogenous steroid 315 hormones on steroid receptors, uterine histological structure and the bacterial 316 flora of the normal bitch. Anim Reprod Sci 1999;56:259-277. 317 18. Grindlay M, Renton JP, Ramsay DH: O-groups of Escherichia coli associated 318 with canine pyometra. Res Vet Sci 1973;14:75-77. 319 118 19. Schoon HA, Schoon D, Nolte I: [The pathogenesis of the "endometritis-pyometra 320 complex" in the female dog]. Zentralbl Veterinarmed A 1992;39:43-56. 321 20. Bjurstrom L: Aerobic bacteria occurring in the vagina of bitches with reproductive 322 disorders. Acta Vet Scand 1993;34:29-34. 323 21. Dhaliwal GK, Wray C, Noakes DE: Uterine bacterial flora and uterine lesions in 324 bitches with cystic endometrial hyperplasia (pyometra). Vet Rec 1998;143:659-325 661. 326 22. Sandholm M, Vasenius H, Kivisto AK. Pathogenesis of canine pyometra. J Am 327 Vet Med Assoc 1975;167:1006-1010. 328 23. Baba E, Hata H, Fukata T, et al: Vaginal and uterine microflora of adult dogs. 329 Am J Vet Res 1983;44:606-609. 330 24. Watts JR, Wright PJ, Whithear KC: Uterine, cervical and vaginal microflora of the 331 normal bitch throughout the reproductive cycle. J Small Anim Pract 1996;37:54-332 60. 333 25. Yuri K, Nakata K, Katae H, et al: Distribution of uropathogenic virulence factors 334 among Escherichia coli strains isolated from dogs and cats. J Vet Med Sci 335 1998;60:287-290. 336 26. Senior DF, deMan P, Svanborg C: Serotype, hemolysin production, and 337 adherence characteristics of strains of Escherichia coli causing urinary tract 338 infection in dogs. Am J Vet Res 1992;53:494-498. 339 27. Feria C, Machado J, Correia JD, et al: Virulence genes and P fimbriae PapA 340 subunit diversity in canine and feline uropathogenic Escherichia coli. Vet 341 Microbiol 2001;82:81-89. 342 119 28. Johnson JR, Stell AL, Delavari P, et al: Phylogenetic and pathotypic similarities 343 between Escherichia coli isolates from urinary tract infections in dogs and 344 extraintestinal infections in humans. J Infect Dis 2001;183:897-906. 345 29. Johnson JR, O'Bryan TT, Low DA, et al: Evidence of commonality between 346 canine and human extraintestinal pathogenic Escherichia coli strains that 347 express papG allele III. Infect Immun 2000;68:3327-3336. 348 30. Wadås B, Kühn I, Lagerstedt AS, et al: Biochemical phenotypes of Escherichia 349 coli in dogs: comparison of isolates isolated from bitches suffering from pyometra 350 and urinary tract infection with isolates from faeces of healthy dogs. Vet Microbiol 351 1996;52:293-300. 352 31. Hagman R, Kindahl H, Fransson BA, et al: Differentiation between pyometra and 353 cystic endometrial hyperplasia/mucometra in bitches by prostaglandin F2-alpha 354 metabolite analysis. Theriogenology 2006;66:198-206. 355 32. Chen YMM, Wright PJ, Lee CS, et al: Uropathogenic virulence factors in isolates 356 of Escherichia coli from clinical cases of canine pyometra and feces of healthy 357 bitches. Vet Microbiol 2003;94:57-69. 358 33. Wullt B, Bergsten G, Samuelsson M, et al: The role of P fimbriae for Escherichia 359 coli establishment and mucosal inflammation in the human urinary tract. Int J 360 Antimicrob Agents 2002;19:522-538. 361 34. Stromberg N, Marklund BI, Lund B, et al: Host-specificity of uropathogenic 362 Escherichia coli depends on differences in binding specificity to Gal alpha 1-363 4Gal-containing isoreceptors. Embo J 1990;9:2001-2010. 364 120 35. Hagberg L, Jodal U, Korhonen TK, et al: Adhesion, hemagglutination, and 365 virulence of Escherichia coli causing urinary tract infections. Infect Immun 366 1981;31:564-570. 367 36. Kreft B, Placzek M, Doehn C, et al: S fimbriae of uropathogenic Escherichia coli 368 bind to primary human renal proximal tubular epithelial cells but do not induce 369 expression of intercellular adhesion molecule 1. Infect Immun 1995;63:3235-370 3238. 371 37. Tiba MR, Yano T, Leite DDS: Genotypic characterization of virulence factors in 372 Escherichia coli strains from patients with cystitis. Revista do Instituto de 373 Medicina Tropical de Sao Paulo 2008;50:255-260. 374 38. Sugiura K, Nishikawa M, Ishiguro K, et al: Effect of ovarian hormones on 375 periodical changes in immune resistance associated with estrous cycle in the 376 beagle bitch. Immunobiology 2004;209:619-627. 377 39. Kolm G, Klein D, Knapp E, et al: Lactoferrin expression in the horse 378 endometrium: relevance in persisting mating-induced endometritis. Vet Immunol 379 Immunopathol 2006;114:159-167. 380 40. Kida K, Baba E, Torii R, et al: Lactoferrin expression in the canine uterus during 381 the estrous cycle and with pyometra. Theriogenology 2006;66:1325-1333. 382 41. Bentwood BJ, Henson PM: The sequential release of granule constituents from 383 human neutrophils. J Immunol 1980;124:855-862. 384 42. Spencer TE, Johnson GA, Bazer FW, et al: Implantation mechanisms: insights 385 from the sheep. Reproduction 2004;128:657-668. 386 121 43. Ishiguro K, Baba E, Torii R, et al: Reduction of mucin-1 gene expression 387 associated with increased Escherichia coli adherence in the canine uterus in the 388 early stage of dioestrus. Vet J 2007;173:325-332. 389 44. Root Kustritz MV: Collection of tissue and culture samples from the canine 390 reproductive tract. Theriogenology 2006;66:567-574. 391 45. Watts JR, Wright PJ, Lee CS, et al: New techniques using transcervical uterine 392 cannulation for the diagnosis of uterine disorders in bitches. J Reprod Fert 393 1997;51(Suppl):283-293. 394 46. Downs M, Miller-Liebl D, Fayrer-Hosken R, et al: Obtaining a useful uterine 395 biopsy specimen in dogs. Vet Med 1994;89:1055-1059. 396 47. Watts JR, Wright PJ, Lee CS: Endometrial cytology of the normal bitch 397 throughout the reproductive cycle. J Small Anim Pract 1998;39:2-9. 398 48. Gunzel-Apel AR, Wilke M, Aupperle H, et al: Development of a technique for 399 transcervical collection of uterine tissue in bitches. J Reprod Fertil Suppl 400 2001;57:61-65. 401 49. LeBlanc MM: 2009. Personal communication. 402 50. Meyers-Wallen VN, Goldschmidt MH, Flickinger GL: Prostaglandin F2 alpha 403 treatment of canine pyometra. J Am Vet Med Assoc 1986;189:1557-1561. 404 51. Gilbert RO, Nothling JO, Oettle EE: A retrospective study of 40 cases of canine 405 pyometra-metritis treated with prostaglandin F-2 alpha and broad-spectrum 406 antibacterial drugs. J Reprod Fertil Suppl 1989;39:225-229. 407 122 52. Verstegen J, Dhaliwal G, Verstegen-Onclin K: Mucometra, cystic endometrial 408 hyperplasia, and pyometra in the bitch: advances in treatment and assessment of 409 future reproductive success. Theriogenology 2008;70:364-374. 410 53. Post K, Evans LE, Jöchle W: Effects of prolactin suppression with cabergoline on 411 the pregnancy of the bitch. Theriogenology 1988;29:1233-1243. 412 54. Onclin K, Verstegen JP: Comparisons of different combinations of analogues of 413 PGF2α and dopamine agonists for the termination of pregnancy in dogs. Vet Rec 414 1999;144:416-419. 415 55. Galac S, Kooistra HS, Butinar J, et al: Termination of mid-gestation pregnancy in 416 bitches with aglepristone, a progesterone receptor antagonist. Theriogenology 417 2000;53:941-950. 418 56. Breitkopf M, Hoffmann B, Bostedt H: Treatment of pyometra (cystic endometrial 419 hyperplasia) in bitches with an antiprogestin. J Reprod Fertil Suppl 1997;51:327-420 331. 421 57. Gobello C, Castex G, Klima L, et al: A study of two protocols combining 422 aglepristone and cloprostenol to treat open cervix pyometra in the bitch. 423 Theriogenology 2003;60:901-908. 424 58. Fieni F: Clinical evaluation of the use of aglepristone, with or without 425 cloprostenol, to treat cystic endometrial hyperplasia-pyometra complex in 426 bitches. Theriogenology 2006;66:1550-1556. 427 59. Soto SM, Smithson A, Horcajada JP, et al: Implication of biofilm formation in the 428 persistence of urinary tract infection caused by uropathogenic Escherichia coli. 429 Clin Microbiol Infect 2006;12:1034-1036. 430 123 60. LeBlanc MM, Magsig J, Stromberg AJ: Use of a low-volume uterine flush for 431 diagnosing endometritis in chronically infertile mares. Theriogenology 432 2007;68:403-412. 433 124 Table 1 Function, classification, and distribution of virulence factors of Escherichia coli isolates from various species. Factor Serogroup or Genes Site Species O Antigens O1, O2, O4, O6, O25 O1, O2, O4, O6, O7, O8, O22, O23, O25, O32, O45, O75, O83, O88 O147 urinary tract [28] uterus [18,21] dog [26,28] human [28] dog [18,21] thermostabile; agglutinating; immunogenic O-specific polysaccharide of the cell wall lipopolysaccharide; possessed by all smooth forms of E. coli type I fimbriae fim (pil) urinary bladder [33] feces [25, 32] uterus [32] dog [25, 32] mouse [33] present on most E. coli; bind cell-bound and secreted mannosylated glycoproteins, Tamm Horsfall protein, and uroplakins of bladder epitheliam; presumed to be able to bind to endometrium P fimbriae papGIII urinary epithelial cells [25,28,33] feces [25,32] uterus [32] human [28] dog [25,28,32] mediate attachment to Galα1→4Galβ-containing glycolipid receptor and coreceptor TLR4 S fimbriae sfa urinary tract [25,28]] uterus[25,32] feces [25,32] dog [25,28,32] humans [25,28] bind eukaryotic glycoproteins with a terminal α-sialic acid; bind laminin and plasminogen; may play a role in penetration of E. coli across basement membrane α-hemolysin hlyA urinary tract [25,28] uterus [32] feces [25,32 dog [25,28,32] human [25,28] common exotoxin; toxic to a wide variety of mammalian cells Cytotoxic Necrotizing Factor 1 cnf1 urinary tract [25,28] uterus [32] feces [25,32] dog [25,28,32] human [25,28] belongs to a group of bacterial necrotic substances; associated with outer membrane vesicles; activates Rho GTPases of host cell leading to macropinocytosis by epithelial cells; may function as a means of entry and survival in epithelial cells Aerobactin and other iron-sequestering systems iuc or aer, fyuA, iutA, iroN urinary tract [25,28] uterus [32] feces [25,32] dog [25,28,32] human [2528] bacterial siderophores (low molecular weight Fe(III)-chelator) 125 << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /All /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Warning /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages false /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness false /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages false /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages false /ColorImageDownsampleType /Average /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages false /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.76 /HSamples [2 1 1 2] /VSamples [2 1 1 2] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages false /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages false /GrayImageDownsampleType /Average /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages false /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.76 /HSamples [2 1 1 2] /VSamples [2 1 1 2] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages false /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages false /MonoImageDownsampleType /Average /MonoImageResolution 300 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages false /MonoImageFilter /FlateEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ENU ([Based on 'No Compression'] [Based on 'No Compression wbleeed'] [Based on '[High Quality Print]'] Use these settings to create Adobe PDF documents for quality printing on desktop printers and proofers. 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