2009: Antimicrobial therapy in bovine reproduction Antimicrobial therapy in bovine reproduction 1 M.A. Edmondson 2 Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, 3 Auburn, AL, USA 4 Abstract 5 The use of antimicrobials, both systemically and locally, has been a hallmark of 6 therapy in the treatment of uterine infections in cattle. However, the use of 7 antimicrobials has not been without controversy regarding their efficacy, effects on future 8 fertility, risk for bacterial resistance and potential residues. This article reviews the 9 immunology and pathophysiology of postpartum uterine infections in cattle and evaluates 10 research regarding the use and efficacy of local and systemic antimicrobial therapies. 11 Keywords: Bovine uterus, metritis, endometritis, antibiotic therapy 12 Introduction 13 The use of antimicrobials has been a conventional therapy in treatment of uterine 14 infections in cattle although the use of antibiotics has not been without controversy. 15 Debate continues regarding antimicrobial efficacy, effects on future fertility, risk for 16 bacterial resistance and residues. The proper use of antimicrobials to treat uterine 17 infections must first begin with an appropriate diagnosis and thorough understanding of 18 the immunology of the uterus, the pathophysiology of uterine infections, and the 19 properties of the various antimicrobial agents that may be used therapeutically. 20 Normal uterine involution 21 Understanding normal uterine involution is vital to understanding and defining 22 postpartum disease in cattle. Lochia are normally present until 14 to 23 days 23 233 postpartum.1 After placental detachment, uterine involution is complete in an average of 24 39 days in normal cows. By day 6 postpartum, caruncle septa are disorganized, and by 25 day 15 caruncles are completely sloughed due to necrosis. By day 26 to 30 postpartum, 26 the surface of the endometrium is covered by new endometrium.2 Cervical involution is 27 slower than uterine involution and by day 15 postpartum, the diameter of the cervix 28 normally exceeds that of the uterine horns. Reported times for gross involution of the 29 uterus and cervix vary from 25 to 47 days. Complete histologic uterine involution takes 30 longer than palpable involution and occurs at 42 to 50 days.1 31 Infection of the bovine uterus 32 The majority of cattle experience bacterial contamination of the uterus at the time 33 of parturition. In the normal cow, the uterus is cleared of this bacterial contamination by 34 four weeks postpartum and when these bacteria are not cleared by the cow’s defense 35 mechanisms, a uterine infection ensues. Numerous bacteria have been isolated from the 36 cow’s postpartum uterus, some of which may be incidental and not cause problems. 37 Uterine infections are most commonly due to Arcanobacterium pyogenes. The gram 38 negative anaerobes Fusobacterium necrophorum and Bacteroides melaninogenicus are 39 frequently associated with A. pyogenes. Other organisms that may be associated with 40 uterine disease in the cow include Pseudomonas aeruginosa, staphylococci, hemolytic 41 streptococci, coliforms, etc. Clostridium sp. may occasionally infect the uterus and cause 42 a severe gangrenous metritis or tetanus. Uterine infections in the cow are associated with 43 retained fetal membranes, dystocia, and delivery of twins.3 44 Metritis is the result of severe inflammation involving all layers of the uterus – 45 endometrial mucosa and submucosa, muscularis, and serosa. Metritis usually develops 46 234 during the first week after calving and is associated with dystocia, retained fetal 47 membranes, and calving trauma. These cattle may be septic and present with fever, 48 depression, and anorexia. A copious amount of fetid vaginal discharge may also be 49 present. Endometritis is characterized by inflammation of the endometrium extending no 50 deeper than the stratum spongiosum. Cows with endometritis are usually not 51 systemically ill, and bacteria are usually eliminated after a few estrous cycles. 52 Endometritis is characterized by a mucopurulent or purulent uterine discharge associated 53 with a chronic uterine infection, usually later than 3 weeks postpartum.1 54 Diagnosing uterine infections 55 Uterine infections are most commonly diagnosed at routine examination of 56 postpartum cows, at breeding, or upon examination of a sick cow. A diagnosis of metritis 57 or endometritis may be made by a variety of methods which include: clinical signs, rectal 58 palpation, vaginoscopy, ultrasonography, uterine culture, uterine cytology, and uterine 59 biopsy. Clinical signs of uterine infection vary with the virulence of the causative 60 organism and the severity of predisposing conditions of the cow. Uterine discharge may 61 vary considerably in color; however, discharges are not usually considered abnormal 62 unless the uterine fluid is fetid or if the cow has clinical signs of sepsis. Palpation per 63 rectum is the most commonly used technique for evaluating the degree of uterine 64 involution prior to breeding and is used to diagnose endometritis through evaluation of 65 the size and consistency of the uterus and cervix and the presence of fluid within the 66 lumen of the uterus. If involution is normal, fluid should not be palpable within the 67 uterine lumen by 14 to 18 days postpartum. In cases of severe metritis, the uterus will be 68 enlarged and friable with occasional adhesions between the uterus and other organs or the 69 235 body wall. Although rectal palpation is commonly used for diagnosis of endometritis, it 70 is neither sensitive nor specific. The evaluation of purulent exudate with the aid of a 71 vaginal speculum may also be a useful tool for diagnosing endometritis. 72 Ultrasonography has also been used to evaluate and characterize intralumenal fluid 73 within the uterus as well as the thickness of the uterine wall. Uterine infections are 74 associated with intrauterine fluid with echogenic particles and a variably thickened 75 uterine wall. Bacterial culture is rarely used as treatment must be initiated before culture 76 results are available. Culture is more commonly used in cases where cows fail to respond 77 to treatment. Endometrial biopsy is not used as commonly in cattle as it is in mares and 78 is reported to have a detrimental effect on future fertility.3,4 79 Acute-phase proteins have been evaluated as markers for endometritis in the 80 postpartum cow. These acute-phase proteins include peripheral blood haptoglobin and 81 α1-acid glycoprotein. Haptoglobin is synthesized in the liver in response to tissue 82 damage and binds free hemoglobin to protect from the oxidative activity of hemoglobin. 83 Serum haptoglobin concentrations increase in dairy cows with acute metritis, but do not 84 increase significantly in cases of chronic metritis.5,6 Another study showed that cows 85 with acute postpartum metritis generally have low concentrations of plasma haptoglobin 86 while cows with severe metritis had consistently higher levels.7 A more recent study 87 indicated that cows with ≥1 g/L of haptoglobin on day 3 postpartum are 6.7 times more 88 likely to develop severe or mild metritis. This study also suggests that an acute phase 89 inflammatory response precedes clinical metritis and that haptoglobin screening may 90 assist in the early detection of metritis.8 The α1-acid glycoprotein has been evaluated as 91 well, but the results were less diagnostic than those for haptoglobin.7 The significance of 92 236 these acute phase proteins in the diagnosis of endometritis or metritis is not fully 93 understood. Intrauterine oxygen reductase potential (Eh) and pH have also been explored 94 as a means to assess the level of bacterial contamination within the lumen of the uterus. 95 The Eh values fell in the presence of infection which created an anaerobic environment 96 within the uterus. It is thought that the drop in Eh is due to either bacterial metabolism or 97 increased oxygen consumption by polymorphonuclear (PMN) cells.9 The pH of uterine 98 discharge collected from cases of endometritis varied from 6.9 to 7.3 which favors the 99 growth of A. pyogenes.10,11 100 Immunology of the bovine uterus 101 The uterine defense mechanisms against contaminating bacteria are maintained in 102 many ways which include 1) anatomically by the simple or pseudostratified columnar 103 epithelium covering the endometrium, 2) chemically by the mucoid secretions from the 104 endometrial glands, and 3) immunologically by PMNs and humoral antibodies.10 105 Disruption of these natural defense mechanisms allows for invasion and colonization of 106 the endometrium by opportunistic pathogens. Inflammation of the bovine endometrium 107 can occur following coitus, artificial insemination, or more commonly postpartum. 108 Uterine cellular immunity 109 In the uterus, the cellular defense against bacterial invaders is provided by uterine 110 leukocytes. The PMN population within the uterine lumen increased after 111 experimentally-induced uterine infections.12,13 At approximately 48 hours postpartum in 112 unassisted calvings, leukocytes begin to accumulate in the uterine lumen along with 113 bacterial contaminants.14 This is the beginning of the normal process of uterine 114 involution. In cases of metritis there is an initial decrease in the phagocytic activity of 115 237 uterine PMNs.10,15 Two to three weeks later when clinical recovery has occurred, the 116 phagocytic activity increases which also coincides with lower numbers of bacteria in the 117 uterine lumen.10,16 118 The cellular immune response of the uterus may be negatively affected by some 119 treatments commonly used to treatment postpartum disorders in the cows.10,17 It has been 120 found that manual removal of fetal membranes, intrauterine antiseptics and disinfectants, 121 and intrauterine antibiotics may inhibit or suppress uterine leukocyte phagocytic activity 122 for several days.10,18 It has also been noted that Lugol’s iodine and polyvinyl-pyrrolidone 123 both cause necrosis of the endometrial epithelium and stimulate uterine defense 124 mechanisms and release of prostaglandin F2α.19-22 Although these agents stimulate 125 uterine defense mechanisms, they also cause endometrial fibrosis and thus should not be 126 used as an intrauterine therapy.10,19,23 127 Elevated blood progesterone concentrations have been found to inhibit both 128 uterine and peripheral blood neutrophil phagocytic activities. The numbers of peripheral 129 blood neutrophils increase slowly from about six weeks prior to parturition and reach a 130 peak on the day of calving.16,24,25 However, maternal and fetal cortisol at the time of 131 calving may suppress neutrophil function.26-28 Immediately postpartum, the phagocytic 132 activity of blood neutrophils declines within the uterine lumen.10,29 The cellular defense 133 mechanisms are preserved by an increase in the number of PMN cells.10,24 During the 134 first three weeks postpartum, the number of peripheral blood PMNs declines and is likely 135 due to the migration of these cells into the mammary gland and uterine lumen.24,30,31 In 136 addition, the phagocytic activity of the PMNs declines which is more marked in older 137 versus younger cows.10,30,31 There is some disagreement as to whether there is a decrease 138 238 in the phagocytic activity of neutrophils in the uterine lumen versus the peripheral 139 blood.32-34 Other leukocytes are present in the endometrium of all animals. Lymphocytes 140 are found within the endometrial epithelium in both cycling and non-cycling ewes and 141 heifers with little variation in numbers at different stages of the estrous cycle.35,36 142 Uterine humoral immunity 143 Protective immunoglobulins have been found in the bovine uterine secretions.37-41 144 Immunoglobulin A (IgA) is produced locally from the mucosa of the bovine uterus 145 whereas IgG is produced from two sites. A portion of IgG1 is produced locally in the 146 endometrium while the remaining IgG1 and all of IgG2 is obtained from peripheral 147 circulation.10,37,42 Experimental uterine infections with pathogenic bacteria have 148 demonstrated immunoglobulins in cervical and vaginal secretions that appear in the order 149 IgM, IgA, and IgG and disappear in the order IgM, IgG, and IgA.10,43 The concentrations 150 of each immunoglobulin depends upon the site of sampling with IgG predominately 151 found in uterine lumen and IgA in the vagina.10,13,43,44 Both IgG and IgM concentrations 152 in lochia from healthy cows fall after calving.45 In cows with postpartum disease, both 153 IgA and IgG concentrations in uterine fluids increase quite rapidly as endometritis 154 develops. However, IgM remains low in cattle with endometritis.46 155 Intrauterine therapy 156 A variety of antibiotics and antiseptics have been infused into the uterus of cows 157 to treat postpartum infections. Intrauterine antimicrobials are used in order to achieve 158 high concentrations at the site of infection but are usually unable to penetrate any deeper 159 than the endometrium.1 The intrauterine use of antimicrobial agents is controversial as 160 some have found intrauterine treatment to be beneficial while others have found these 161 239 agents to have no effect or a detrimental effect. The bovine uterus is an anaerobic 162 environment. Thus, antibiotics that are chosen for intrauterine infusion must be active in 163 the absence of oxygen. Additionally, most antibiotics depress the activity of uterine 164 neutrophils and interfere with uterine defense mechanisms.3 Thus, one must carefully 165 evaluate the evidence regarding intrauterine antimicrobial use and carefully consider both 166 the advantages and disadvantages associated with therapy. 167 Historically, intrauterine use of antimicrobials has been a common therapy for 168 treatment of uterine infections. Antimicrobials reportedly used for uterine infections 169 include tetracycline, penicillin, cephapirin, chloramphenicol, Lugol’s iodine, gentamycin, 170 spectinomycin, sulfonamides, nitrofurasone, povidone iodine solution, urea, and 171 chlorhexidine.1 Most of these compounds are not approved for intrauterine use and have 172 no published withdrawal times. There are also reports that intrauterine infusion of 173 antibiotics cause drug residues in milk.47,48 Additionally, regulatory guidelines must be 174 adhered to for extralabel use of antimicrobials in food animals. Intrauterine therapy is 175 considered an extralabel use, and thus may be prohibited for many antibiotics, 176 particularly in the United States. 177 The organisms that cause most postpartum infections are usually sensitive to 178 penicillin. However, bacterial contaminants present within the uterus during the first 179 several weeks postpartum produce penicillinase which makes penicillin useless if used 180 locally in the early (less than 30 days) postpartum period. By 30 days postpartum, the 181 contaminating bacteria are usually eliminated and intrauterine treatment with penicillin is 182 more likely to be effective.3 Other factors may also affect the efficacy of intrauterine 183 240 antibiotic therapy. Uterine lochia present during uterine infections contain organic fluids 184 and debris that can render certain antibiotics, such as sulfonamides, ineffective. 185 More recently, oxytetracycline has been the antimicrobial commonly used for 186 intrauterine therapy.3 However, one study indicated that most isolates of A. pyogenes are 187 resistant to oxytetracycline. This study also showed that large doses of intrauterine 188 oxytetracycline did not affect the frequency of isolation of A. pyogenes.3,49 In addition, 189 oxytetracycline and Lugol’s iodine are quite irritating and are reported to cause 190 coagulation necrosis of the endometrium.3,50 Although some studies indicate improved 191 reproductive performance with the use of intrauterine oxytetracycline, it has been 192 speculated that this improvement may be due to local prostaglandin production due to 193 chemical irritation of the endometrium.51 194 In general, intrauterine infusion of antimicrobials has failed to show any increase 195 in reproductive performance.1 Two large field studies evaluated the use of cephapirin 196 benzoate in cows with clinical endometritis and demonstrated an improvement in 197 reproductive performance.52-54 But other studies have shown no improvement in 198 reproductive performance when evaluating intrauterine administration of cephapirin 199 benzoate.53 The appropriate use of intrauterine antibiotics to treat uterine infections still 200 remains controversial as only a limited number of studies indicate the efficacious use of 201 intrauterine antibiotics. 202 Numerous antiseptics have been used to lavage the postpartum bovine uterus with 203 iodine and chlorhexidine solutions being most commonly used. Many of these solutions 204 are quite irritating to the endometrium and are thought to stimulate endogenous 205 prostaglandin release. One study showed that the incidence of retained fetal membranes 206 241 and endometritis was reduced in cows that received 500 mL of 2% Lugol’s iodine 207 immediately after calving and again 6 hours later. However, this study did not evaluate 208 the future reproductive performance of these treated cows.3,55 Another study evaluated 209 the use of 50 to 100 mL of 2% povidone iodine solution in the uterus one month 210 postpartum and found that the reproductive performance of normal cows was not 211 improved and that the treatment was detrimental to the fertility of cows with 212 endometritis.3,23 213 Systemic antibiotic therapy 214 Cattle with metritis often suffer moderate to severe illness. These cattle are often 215 septic with fever, depression, and anorexia. A variety of antibiotics have been 216 recommended for parenteral use in cattle suffering from uterine infections. Penicillin or 217 one of the synthetic penicillin analogs and ceftiofur are among the more common 218 antibiotics used systemically in cattle suffering from metritis. Systemic use of 219 oxytetracyline may not be efficacious because of the difficulty in achieving the minimal 220 inhibitory concentration (MIC) required for A. pyogenes in the uterine lumen.3 However, 221 one study found clinical improvement of cattle suffering from metritis with the use of 222 tetracycline at 10mg/kg.56 223 Ceftiofur is a third-generation cephalosporin that has broad-spectrum activity 224 against gram negative and gram positive bacteria.57 Ceftiofur is reported to reach all 225 layers of the uterus without causing violative residues in milk. Ceftiofur is approved in 226 the United States for systemic administration to lactating cows affected with metritis.3 A 227 subcutaneous dose of ceftiofur at 1mg/kg in postpartum cows results in a concentration of 228 ceftiofur and its active metabolites in plasma, uterine tissues, and lochia higher than the 229 242 MIC for most of the common pathogens involved in metritis.58 One study demonstrated 230 that ceftiofur administered at 2.2mg/kg daily for five days was effective in treating cows 231 with metritis.57 Another study supported these findings and showed ceftiofur 232 administered at 2.2 mg/kg once daily for five days is as effective for treating metritis as 233 procaine penicillin G or procaine penicillin G with intrauterine infusion of 234 oxytetracycline.59 235 Because of the reported lack of efficacy and potential detrimental effects of future 236 fertility, intrauterine infusion of antibiotics is not a favored treatment for most cases of 237 metritis. Certain systemic antibiotics have demonstrated their effectiveness at treating 238 uterine infections in cattle. Thus, most cases of metritis, especially cows that are toxic, 239 should be treated with systemic antibiotics such as penicillin or ceftiofur. 240 Conclusion 241 There are no antibiotics currently approved for intrauterine administration in the 242 United States. Intrauterine infusion of antibiotics leads to contamination in milk and 243 tissues for which appropriate withdrawal times have not been ascertained. In addition, 244 the assays used on farms to detect antibiotics in milk may not be accurate. Although 245 some studies indicate a positive response to therapy with the use of intrauterine 246 antimicrobials, most studies do not show an improvement in reproductive performance or 247 clinical signs of disease when comparing intrauterine antimicrobial therapy and systemic 248 antibiotic therapy. 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