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Mycoplasma infection in the uterus of early postpartum dairy cows and its relation to dystocia and endometritis
Theriogenology 79 (2013) 180–185
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Theriogenology journal homepage: www.theriojournal.com
Mycoplasma infection in the uterus of early postpartum dairy cows and its relation to dystocia and endometritis Mohamed Elshabrawy Ghanem a, b, Hidetoshi Higuchi c, Erisa Tezuka a, Hideki Ito a, Bhuminand Devkota a, Yoshiaki Izaike a, Takeshi Osawa a, *,1 a b c
Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Morioka, Japan Department of Theriogenology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt Animal Health Laboratory, Faculty of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 March 2012 Received in revised form 31 August 2012 Accepted 27 September 2012
This study investigated the incidence of mycoplasma infection in the uterus of postpartum Holstein dairy cows and its relationship to the occurrence of endometritis. The genital tracts of 209 cows from three dairy farms in the Iwate Prefecture, Japan, were examined at Weeks 5 and 7 postpartum. The condition of the cervicovaginal mucus was assessed using a Metricheck device and assigned a score from 0 (clear mucus) to 4 (purulent material with fetid odor). Intrauterine samples (N ¼ 418) were collected at Weeks 5 and 7 postpartum using a cytobrush. After its withdrawal, swab samples were placed in mycoplasma culture broth at 37 C for 72 hours. A novel and rapid polymerase chain reaction was used to detect seven mycoplasma species (Mycoplasma bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens, and M. canadense). The cytobrush was also rolled gently along the length of a glass slide for subsequent polymorphonuclear neutrophil count. The diagnostic criteria for cytological endometritis were 6% or more and 4% or more polymorphonuclear neutrophils at Weeks 5 and 7, respectively. From a subset of cows, additional swabs were rolled against the cytobrush and then placed in transport medium. These samples were then plated on specific agar plates and cultured under aerobic and anaerobic conditions to identify other bacteria present. The incidence of dystocia at the last calving was compared in mycoplasma positive and negative cows. Of the seven mycoplasma species, only M. bovigenitalium was detected; it was detected in 31 of the 418 uterine swabs (7.4%). Twenty-four cows were positive for M. bovigenitalium (eight cows at Week 5, nine cows at Week 7, and seven cows at both Weeks 5 and 7). The incidence of dystocia was higher (P < 0.0001) in mycoplasma positive (7/24; 29.2%) compared with mycoplasma negative (4/185; 2.2%) cows. However, there was no significant association between dystocia at last calving and subsequent uterine infection with other bacteria. In addition, the incidence of cytologic endometritis was higher (P < 0.05) in mycoplasma positive (8/16; 50%) than in mycoplasma negative (47/193; 24.4%) cows at Week 7. Therefore, we concluded that M. bovigenitalium infection in the uterus might be associated with recent dystocia and with cytologic endometritis in postpartum dairy cows. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Mycoplasma Uterus Endometritis Dystocia Dairy cows
* Corresponding author. Tel.: þ81-985-58-7787; fax: þ81-985-58-7787. E-mail address: [email protected] (T. Osawa). 1 Present address: Laboratory of Theriogenology, Department of Veterinary Sciences, University of Miyazaki, Gakuen-kibanadai-nishi 1-1 Miyazaki 889-2192, Japan. 0093-691X/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2012.09.027
1. Introduction Heavy bacterial colonization subsequent to trauma, dystocia, or poor hygiene and poor uterine defense mechanisms can lead to establishment of puerperal uterine
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infection [1–4]. In cattle, nonpathogenic bacteria in the uterus disappear more quickly after a difficult calving than after normal parturition, and pathogenic isolates persist longer in dystocia-affected animals [5]. The presence of pathogenic bacteria in the uterus causes inflammation and histologic lesions in the endometrium, delays uterine involution, and hinders embryo survival [6]. After the first isolation of Mycoplasma bovigenitalium in 1947, numerous studies throughout the world have demonstrated the presence of this organism in the genital tracts of healthy and diseased cows and bulls [7,8]. Although the experimental pathogenicity of some isolates has been demonstrated, there is still doubt regarding the exact role of this organism in bovine reproductive disease [9]. M. bovigenitalium has been detected in the vaginal mucus of normal and repeat breeder cows, which has led to speculation concerning its role as a pathogen [10]. It has been found in cows with low fertility in which no other cause of infertility was identified [11]. M. bovigenitalium has been implicated in several outbreaks of granulopapular vulvovaginitis, which is characterized by lesions that resemble ‘wolf bites’ on the external vaginal labia and granulopustular lesions of the vagina [12]. In South Africa, M. bovigenitalium was recovered from the cervicovaginal mucus of 47% of gynecologically normal postpartum cows [13]. However, there is little information available regarding isolation of M. bovigenitalium from the uterus of live cows. Additionally, there has been little reported association between mycoplasma infection and cytological endometritis based on polymorphonuclear neutrophil (PMN) percentage. The percentage of PMN in the total number of endometrial cells is considered indicative of cytologic endometritis in cows, with 6% or more PMNs at the first reproductive exam (35 3 days) and 4% or more PMNs at the second (56 3 days) postpartum reproductive exam resulting in a diagnosis of endometritis [14]. Therefore, the objectives of the present study were to investigate the incidence of mycoplasma infection in the uterus of postpartum cows and to examine the association of mycoplasma infection with calving conditions and cytologic endometritis.
2. Materials and methods 2.1. Animals Holstein cows (N ¼ 209) from three herds in the Iwate Prefecture, Japan, were used in this study. All cows were housed in free stalls, fed an identical diet formulated according to standard guidelines, and milked twice daily. The average parity and age of the cows were 1.4 0.1 and 3.6 0.1 years, respectively. The ease of the previous calving (eutocia or dystocia) was recorded for all cows. Dystocia was defined as any calving that required an intervention to assist the cow. To determine whether dystocia was a predisposing factor for bacterial infections of the postpartum uterus, the incidence of dystocia in a subset of cows (N ¼ 88) with or without postpartum uterine bacterial infection was calculated. The study period was from June 2011 to February 2012.
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2.2. Evaluation of cervicovaginal mucus using a Metricheck device Before examination, the vulva was cleaned with paper towels. Cows were examined using a Metricheck device (Simcro Tech Ltd., Hamilton, New Zealand) at Week 5 (34.9 0.2 days) and Week 7 (48.6 0.2 days) postpartum. Cervicovaginal mucus was collected as described [15]. The material within the concave surface of the device and ⁄or adherent to the convex surface was visually examined and scored on a 0 to 4 scale as follows: 0 ¼ clear mucus; 1 ¼mucus with flecks of pus; 2 ¼ mucopurulent discharge; 3 ¼ purulent discharge; and 4 ¼ foul smelling discharge [16,17]. 2.3. Endometrial cytology A cytological sample of the endometrium from the uterine body was collected at Weeks 5 and 7 using a cytobrush (Puritan Medical Products Company L.L.C., Guilford, ME, USA) adapted for use in cattle [17,18]. The brush was retracted into a stainless steel tube before removal from the uterus. Slides for cytologic examination were prepared on farm by rolling the cytobrush on a clean glass microscope slide and fixing with cytofixative (Cytokeep II, Alfresa Pharma Corporation, Osaka, Japan). Fixed slides were stained with Diff-Quik (Sysmex, Kobe, Japan) for 20 seconds, washed in distilled water, and dried. A quantitative cytologic assessment of endometrial inflammation was carried out by counting a minimum of 200 endometrial cells at magnification 400 and determining the percentage of PMNs within those cells. The diagnostic criteria for cytologic endometritis were 6% or more and 4% or more PMNs, at postpartum Weeks 5 and 7, respectively [14]. 2.4. Mycoplasma culture techniques After the withdrawal of the cytobrush from the uterus and before rolling it on the glass slide, swab samples were taken, placed in mycoplasma culture broth (Kanto Kagaku, Japan), and incubated at 37 C for 72 hours. Then, 100 mL of the broth culture was plated on a mycoplasma agar plate (Kanto Kagaku, Japan) and incubated in 5% CO2 at 37 C for 3 to 14 days to produce typical mycoplasma colonies. If typical mycoplasma colonies appeared within 3 days, species identification was performed using polymerase chain reaction (PCR). However, if no typical mycoplasma colonies grew during the first 3 days of incubation, the culture was continued for a total of 14 days to ensure that the sample was mycoplasma negative (this was subsequently confirmed using PCR). Each broth culture was analyzed using the simplified PCR procedure described below. 2.5. Detection and identification of mycoplasma species by PCR A novel and rapid PCR was used to detect seven mycoplasma species (Mycoplasma bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens, and M. canadense) [19]. A simplified PCR was performed in
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a total reaction volume of 20 mL in an iCycler PCR System (Bio-Rad Laboratories Inc., Richmond, CA, USA). Conditions for the simplified PCR were as follows: initial denaturation at 95 C for 10 minutes followed by 35 cycles of denaturation at 94 C for 30 seconds, annealing at 60 C for 45 seconds, and extension at 72 C for 1 minute. The PCR products were separated by electrophoresis on 1.5% (wt/vol) agarose gels, stained with ethidium bromide, and visualized with an ultraviolet transilluminator. 2.6. Aerobic and anaerobic bacterial culture For a subset of cows (N ¼ 88), uterine swab samples were collected using the cytobrush technique at Weeks 5 and 7. Sterilized swabs were rolled against the cytobrush and then placed in transport medium on the farm and transported to the laboratory. The samples were then plated on a blood agar plate, a chocolate agar plate, and a deoxycholate-hydrogen sulfide-lactose agar plate, and incubated at 35 1 C under aerobic conditions. The samples were also plated on a brain-heart infusion agar plate and incubated at 35 1 C under anaerobic conditions. Colonies present at 24 and 48 hours (aerobic conditions) or at 48 hours (anaerobic conditions) were harvested. When no growth was observed by 48 hours under either aerobic or anaerobic conditions, the samples were plated onto Gifu anaerobic medium semisolid medium and incubated at 35 1 C under aerobic conditions. Bacterial growth was monitored at 24, 48, and 72 hours. If growth was observed on the surface of the medium, the bacteria were extracted from the Gifu anaerobic medium onto plate media and incubated under aerobic conditions. If growth was observed in the deeper part of the media, the extracted bacteria were plated and incubated under anaerobic conditions. Bacterial species were identified by colony morphology, Gram stain, and biochemical characteristics [20]. On the basis of the presence or absence of growth of any bacterial isolates from the uterine samples, cows were considered positive or negative for bacterial infection of the uterus. 2.7. Statistical analysis The incidence of cytologic endometritis and dystocia at the last calving was compared in mycoplasma positive and negative cows using Fisher’s exact probability test. The incidence of dystocia was compared in a subset of cows with uterine bacterial infections using Fisher’s exact probability test. In mycoplasma positive cows, differences between the PMN percentages at Weeks 5 and 7 in cows with and without a history of dystocia at last calving were determined by Student’s t test. Data are presented as mean SEM. Probability values less than 0.05 were considered significant. 3. Results 3.1. Incidence of mycoplasma infection in postpartum cows In total, 418 uterine swabs were collected from 209 cows at Weeks 5 and 7 postpartum. Of these, 31 (7.4%) swabs (15 at
Fig. 1. Molecular diagnosis of Mycoplasma species by polymerase chain reaction, showing the detection of only M. bovigenitalium (450 base pairs [bp]) in Holstein cows. Lane (M), marker of 100 bp; lane (1), uterine isolate of M. bovigenitalium; lane (2), positive control isolate of M. bovigenitalium.
Week 5 and 16 at Week 7) were confirmed positive for M. bovigenitalium (Fig. 1). The mycoplasma positive uterine swabs were collected from 24 cows (eight cows at Week 5, nine at Week 7, and seven at both Weeks 5 and 7). Of the 24 cows infected with M. bovigenitalium, seven (29.2%) had calved during the summer, and the remaining cows had calved from September to December. In addition, 50% of the mycoplasma positive cows were at their first parity. The other six species of mycoplasma were not detected in the uteri of any postpartum cows in this study. 3.2. Incidence of dystocia in mycoplasma positive and negative cows The incidence of dystocia at last calving was higher (P < 0.0001) in mycoplasma positive (29.2%; 7/24) compared with mycoplasma negative (2.2%; 4/185) cows (Table 1). In contrast, the incidence of dystocia at the last calving was not different between cows that cultured positive and negative for other bacteria (P ¼ 0.64; Table 2).
M.E. Ghanem et al. / Theriogenology 79 (2013) 180–185 Table 1 Incidence of dystocia at last calving in relation to M. bovigenitalium infection in Holstein cows. M. bovigenitalium infection
Normal calving
Dystocia
Total
Incidence (%)
P
Negative Positive Total
181 17 198
4 7 11
185 24 209
2.2 29.2 d
<0.0001 d d
3.3. Mycoplasma infection in relation to cytologic endometritis At Week 5, there was no difference in the incidence of cytologic endometritis in mycoplasma positive and mycoplasma negative cows (P ¼ 0.2). However, at Week 7, the incidence of cytologic endometritis was higher (P < 0.05) in mycoplasma positive (50%; 8/16) compared with mycoplasma negative (24.4%; 47/193) cows (Table 3). 3.4. Relationship between dystocia at last calving and cytologic endometritis in mycoplasma positive cows Out of the 17 mycoplasma positive cows that did not experience dystocia at their last calving, five (29.4%) had cytological endometritis. However, five of the seven cows (71.4%) with a history of dystocia at last calving had cytologic endometritis. In mycoplasma positive cows, the percentage of PMN at Week 5 did not differ (P ¼ 0.12) between cows with a history of dystocia at last calving (12.5 9.5) and cows without dystocia (2.6 1.1). However, at Week, 7 cows with a history of dystocia at last calving (12.3 4.7) had a greater PMN score (P < 0.05) compared with cows without dystocia (4.4 1.5). 4. Discussion One of the objectives of this study was to evaluate the incidence of uterine mycoplasma infections. Although it is widely accepted that mycoplasma might cause a variety of clinical symptoms in cattle, the prevalence and reproductive effect of mycoplasma infections in Holstein cows are not well established. In this study, M. bovigenitalium was detected in 7.4% of postpartum Holstein cows. The reported rate of M. bovigenitalium isolated from cervicovaginal mucus samples was 11% in healthy cows [21]. Higher (although not usually significant) isolation rates have been obtained from infertile cows [22], and those with granular vulvitis [12,23]. Moreover, the results of attempts to culture M. bovigenitalium from samples taken from infertile cows
Table 2 Incidence of dystocia at last calving in relation to postpartum uterine bacterial infection in Holstein cows. Postpartum uterine bacterial infection
Normal calving
Dystocia
Total
Incidence (%)
P
Negative Positive Total
35 48 83
1 4 5
36 52 88
2.8 7.7 d
0.64 d d
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have been inconclusive. In one study, only three uteri of 80 abattoir-derived samples from infertile beef cows were positive for mycoplasma: a mixed culture of M. bovigenitalium and M. arginini was recovered from one specimen; M. bovigenitalium in pure culture was recovered from two specimens; and M. bovigenitalium was also isolated from the cotyledons and kidney of one fetus [7]. It is difficult to compare the prevalence of M. bovigenitalium between reports, because of differences in sampling sites and diagnostic methods. However, when comparing the prevalence rate with other studies that examined the cervicovaginal mucus samples, the prevalence of M. bovigenitalium in this study seemed lower than previously reported in healthy animals [13,21], and higher than previously reported in infertile cows [7]. In this study, mycoplasma was obtained from the uterus of postpartum Holstein cows using a cytobrush technique. To the best of our knowledge, this was the first study that has isolated mycoplasma directly from the uterus of living cows. Only a few reports of the isolation of mycoplasma from uteri obtained at slaughter are available [7]. M. bovigenitalium has been recovered from the uterine secretions of cows with breeding problems and endometritis by transrectal massage of the uterine horns [24]. Additionally, M. bovigenitalium was isolated from the cervicovaginal secretions of a cow with pyometra. However, there is considerable doubt that this isolation was truly from the uterus, because the cervical canal was open and the uterus was greatly distended with mucopurulent fluid [10]. There is no available literature on the relationship between dystocia and mycoplasma infection in the uterus of postpartum cows. In the present study, there was a strong positive association between mycoplasma infection and dystocia at last calving. In that regard, nearly 30% of the cows with M. bovigenitalium infection had a history of dystocia. This might be because of the entrance of microbes into the uterus at calving, or to reduced immune function in the genital tracts of cows that have recently experienced dystocia. Interestingly, dystocia was not associated with subsequent postpartum uterine bacterial infection in a subset of cows in the current study. Further investigation is required to elucidate the factors underlying uterine infections, especially in cows with a history of dystocia. After normal parturition the uterus harbors a wide range of microbes; this is mainly attributed to aspiration of environmental contaminants because of the negative uterine pressure created after expulsion of the calf [25]. However, the presence of these microbes does not necessarily indicate infection, and they are usually cleared by effective local and general defense mechanisms in the weeks after calving [26]. However, after an abnormal calving, the microbes might colonize the endometrium and establish infection, leading to puerperal metritis [27]. Prolonged dystocia, uterine inertia, and/or retained fetal membranes can result in the cervix remaining open for a longer interval, thereby providing greater opportunity for bacterial invasion [28,29]. In addition, the likelihood of uterine contamination increases in dystocia cases during the process of examination and assisted delivery of fetus [27]. The bacterial count increases by 12 to 14 hours after birth in bovine dystocia cases [28]. Under normal
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Table 3 Incidence of cytological endometritis at W5 and W7 postpartum in relation to M. bovigenitalium infection in Holstein cows. M. bovigenitalium infection
W5
W7 a
Endometritis
Negative Positive Total
Without
With
138 13 151
56 2 58
Total
194 15 209
Incidence (%)
28.9 13.3
P
0.2
Endometritisa Without
With
146 8 154
47 8 55
Total
Incidence (%)
P
193 16 209
24.4 50.0
0.03
a Diagnostic criteria for cytological endometritis were 6% or more and 4% or more polymorphonuclear neutrophils at Weeks 5 (W5) and 7 (W7), respectively.
circumstances, contaminating organisms are eliminated from the uterus through phagocytosis by migrating leukocytes and through physical expulsion because of uterine contractions. Most of the mycoplasma-positive cows in the present study had a history of dystocia in the summer season. The highest incidence of dystocia was recorded in primipara [30]. There were no statistically significant differences in the rate of occurrence of dystocia in different seasons, but numerically more cases were recorded in the summer, perhaps because of thermal stress [31]. In the present study, some cows were mycoplasma positive at Week 5 and negative at Week 7, suggesting that the uterine immune response eliminated the mycoplasma infection. Other cows were mycoplasma negative at Week 5 and positive at Week 7; this development of infection between Weeks 5 and 7 might be because of a decline in the immune status of the animals. In contrast, it has been previously reported that most cows that are negative for subclinical endometritis at Week 4 postpartum are also negative at Week 8. Similarly, a higher proportion of cows that have subclinical endometritis at Week 4 are also positive at Week 8 postpartum, indicating that subclinical endometritis can persist for a prolonged interval and/or that the same animal can be reinfected on different occasions, although this requires further study [32]. To the best of our knowledge, there are no reports in the literature that describe the persistence of M. bovigenitalium in the bovine uterus. However, mammary infection with M. bovis has been reported to last for varying intervals, from less than 30 days in some cases, to the end of the cow’s lactation in others. An intramammary infection with a high somatic cell count because of M. bovis persisted in some cows for times ranging from 7 weeks to more than 5 months, even after they had returned to apparently normal milk production [33]. These results, like the repeated reisolations of M. bovis over extended intervals from naturally occurring cases of mastitis [34], provided evidence that M. bovis can establish a persistent infection in the bovine mammary gland and that it might cause an extended period of subclinical mastitis after a variable period of clinical M. bovis mastitis [33]. In vitro studies using endometrial cells infected with M. bovigenitalium might provide clues regarding factors associated with the persistence of the organism in the uterine cavity. Little information exists on the relationship between mycoplasma infection and cytological endometritis. One of the novel findings in this study was that the percentage of PMN cells was greater in M. bovigenitalium-positive cows
with a history of dystocia at the last calving than in cows without dystocia. Perhaps M. bovigenitalium aggravated the severity of endometritis when it was associated with dystocia. Furthermore, M. bovigenitalium seemed to be pathogenic to the endometrium and not an opportunistic organism. However, additional investigations in a large population of postpartum cows are required to confirm this. There was no significant association between recent dystocia and uterine infection with bacteria other than mycoplasma. This might be because of sampling technique; we used a cytobrush to collect samples from the uterine body for bacterial culture. Although previous studies have shown that the sensitivity of cytobrush cytology for diagnosing subclinical endometritis is high [18], it is possible that some pathogens cannot be isolated from samples collected in this manner. It has been previously reported that, even in samples with cytologic evidence of inflammation and phagocytized bacteria, culture of uterine biopsies taken at the same time might be negative for bacteria. Consequently, even bacteriological investigation of uterine biopsies did not detect all cases of infection [35]. In addition, the cytobrush might touch only a small area of the endometrium and focal infections might be missed [36]. In this study, we did not examine or sample the animals after Week 7 postpartum. Future studies should focus on the follow-up of the infected cows to evaluate the pathogenicity of mycoplasma, the ability of cows to self-cure the infection, and the subsequent reproductive performance of infected animals. In addition, the significance of M. bovigenitalium infection in other places in the body besides the uterus should be evaluated. 4.1. Conclusions Out of seven species of mycoplasma, only M. bovigenitalium was detected in the uterus of postpartum cows. M. bovigenitalium infection has an incidence of 7.4% and might be associated with dystocia at last calving and with cytological endometritis in postpartum dairy cows. Acknowledgments M. E. Ghanem is supported by a Japan Society for the Promotion of Science (JSPS) fellowship. The authors thank Miyarisan Pharmaceutical Co. Ltd., for providing mycoplasma culture broth, and the members of the Food
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Mycoplasma infection in the uterus of early postpartum dairy cows and its relation to dystocia and endometritis Mohamed Elshabrawy Ghanem a, b, Hidetoshi Higuchi c, Erisa Tezuka a, Hideki Ito a, Bhuminand Devkota a, Yoshiaki Izaike a, Takeshi Osawa a, *,1 a b c
Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Morioka, Japan Department of Theriogenology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt Animal Health Laboratory, Faculty of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 March 2012 Received in revised form 31 August 2012 Accepted 27 September 2012
This study investigated the incidence of mycoplasma infection in the uterus of postpartum Holstein dairy cows and its relationship to the occurrence of endometritis. The genital tracts of 209 cows from three dairy farms in the Iwate Prefecture, Japan, were examined at Weeks 5 and 7 postpartum. The condition of the cervicovaginal mucus was assessed using a Metricheck device and assigned a score from 0 (clear mucus) to 4 (purulent material with fetid odor). Intrauterine samples (N ¼ 418) were collected at Weeks 5 and 7 postpartum using a cytobrush. After its withdrawal, swab samples were placed in mycoplasma culture broth at 37 C for 72 hours. A novel and rapid polymerase chain reaction was used to detect seven mycoplasma species (Mycoplasma bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens, and M. canadense). The cytobrush was also rolled gently along the length of a glass slide for subsequent polymorphonuclear neutrophil count. The diagnostic criteria for cytological endometritis were 6% or more and 4% or more polymorphonuclear neutrophils at Weeks 5 and 7, respectively. From a subset of cows, additional swabs were rolled against the cytobrush and then placed in transport medium. These samples were then plated on specific agar plates and cultured under aerobic and anaerobic conditions to identify other bacteria present. The incidence of dystocia at the last calving was compared in mycoplasma positive and negative cows. Of the seven mycoplasma species, only M. bovigenitalium was detected; it was detected in 31 of the 418 uterine swabs (7.4%). Twenty-four cows were positive for M. bovigenitalium (eight cows at Week 5, nine cows at Week 7, and seven cows at both Weeks 5 and 7). The incidence of dystocia was higher (P < 0.0001) in mycoplasma positive (7/24; 29.2%) compared with mycoplasma negative (4/185; 2.2%) cows. However, there was no significant association between dystocia at last calving and subsequent uterine infection with other bacteria. In addition, the incidence of cytologic endometritis was higher (P < 0.05) in mycoplasma positive (8/16; 50%) than in mycoplasma negative (47/193; 24.4%) cows at Week 7. Therefore, we concluded that M. bovigenitalium infection in the uterus might be associated with recent dystocia and with cytologic endometritis in postpartum dairy cows. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Mycoplasma Uterus Endometritis Dystocia Dairy cows
* Corresponding author. Tel.: þ81-985-58-7787; fax: þ81-985-58-7787. E-mail address: [email protected] (T. Osawa). 1 Present address: Laboratory of Theriogenology, Department of Veterinary Sciences, University of Miyazaki, Gakuen-kibanadai-nishi 1-1 Miyazaki 889-2192, Japan. 0093-691X/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2012.09.027
1. Introduction Heavy bacterial colonization subsequent to trauma, dystocia, or poor hygiene and poor uterine defense mechanisms can lead to establishment of puerperal uterine
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infection [1–4]. In cattle, nonpathogenic bacteria in the uterus disappear more quickly after a difficult calving than after normal parturition, and pathogenic isolates persist longer in dystocia-affected animals [5]. The presence of pathogenic bacteria in the uterus causes inflammation and histologic lesions in the endometrium, delays uterine involution, and hinders embryo survival [6]. After the first isolation of Mycoplasma bovigenitalium in 1947, numerous studies throughout the world have demonstrated the presence of this organism in the genital tracts of healthy and diseased cows and bulls [7,8]. Although the experimental pathogenicity of some isolates has been demonstrated, there is still doubt regarding the exact role of this organism in bovine reproductive disease [9]. M. bovigenitalium has been detected in the vaginal mucus of normal and repeat breeder cows, which has led to speculation concerning its role as a pathogen [10]. It has been found in cows with low fertility in which no other cause of infertility was identified [11]. M. bovigenitalium has been implicated in several outbreaks of granulopapular vulvovaginitis, which is characterized by lesions that resemble ‘wolf bites’ on the external vaginal labia and granulopustular lesions of the vagina [12]. In South Africa, M. bovigenitalium was recovered from the cervicovaginal mucus of 47% of gynecologically normal postpartum cows [13]. However, there is little information available regarding isolation of M. bovigenitalium from the uterus of live cows. Additionally, there has been little reported association between mycoplasma infection and cytological endometritis based on polymorphonuclear neutrophil (PMN) percentage. The percentage of PMN in the total number of endometrial cells is considered indicative of cytologic endometritis in cows, with 6% or more PMNs at the first reproductive exam (35 3 days) and 4% or more PMNs at the second (56 3 days) postpartum reproductive exam resulting in a diagnosis of endometritis [14]. Therefore, the objectives of the present study were to investigate the incidence of mycoplasma infection in the uterus of postpartum cows and to examine the association of mycoplasma infection with calving conditions and cytologic endometritis.
2. Materials and methods 2.1. Animals Holstein cows (N ¼ 209) from three herds in the Iwate Prefecture, Japan, were used in this study. All cows were housed in free stalls, fed an identical diet formulated according to standard guidelines, and milked twice daily. The average parity and age of the cows were 1.4 0.1 and 3.6 0.1 years, respectively. The ease of the previous calving (eutocia or dystocia) was recorded for all cows. Dystocia was defined as any calving that required an intervention to assist the cow. To determine whether dystocia was a predisposing factor for bacterial infections of the postpartum uterus, the incidence of dystocia in a subset of cows (N ¼ 88) with or without postpartum uterine bacterial infection was calculated. The study period was from June 2011 to February 2012.
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2.2. Evaluation of cervicovaginal mucus using a Metricheck device Before examination, the vulva was cleaned with paper towels. Cows were examined using a Metricheck device (Simcro Tech Ltd., Hamilton, New Zealand) at Week 5 (34.9 0.2 days) and Week 7 (48.6 0.2 days) postpartum. Cervicovaginal mucus was collected as described [15]. The material within the concave surface of the device and ⁄or adherent to the convex surface was visually examined and scored on a 0 to 4 scale as follows: 0 ¼ clear mucus; 1 ¼mucus with flecks of pus; 2 ¼ mucopurulent discharge; 3 ¼ purulent discharge; and 4 ¼ foul smelling discharge [16,17]. 2.3. Endometrial cytology A cytological sample of the endometrium from the uterine body was collected at Weeks 5 and 7 using a cytobrush (Puritan Medical Products Company L.L.C., Guilford, ME, USA) adapted for use in cattle [17,18]. The brush was retracted into a stainless steel tube before removal from the uterus. Slides for cytologic examination were prepared on farm by rolling the cytobrush on a clean glass microscope slide and fixing with cytofixative (Cytokeep II, Alfresa Pharma Corporation, Osaka, Japan). Fixed slides were stained with Diff-Quik (Sysmex, Kobe, Japan) for 20 seconds, washed in distilled water, and dried. A quantitative cytologic assessment of endometrial inflammation was carried out by counting a minimum of 200 endometrial cells at magnification 400 and determining the percentage of PMNs within those cells. The diagnostic criteria for cytologic endometritis were 6% or more and 4% or more PMNs, at postpartum Weeks 5 and 7, respectively [14]. 2.4. Mycoplasma culture techniques After the withdrawal of the cytobrush from the uterus and before rolling it on the glass slide, swab samples were taken, placed in mycoplasma culture broth (Kanto Kagaku, Japan), and incubated at 37 C for 72 hours. Then, 100 mL of the broth culture was plated on a mycoplasma agar plate (Kanto Kagaku, Japan) and incubated in 5% CO2 at 37 C for 3 to 14 days to produce typical mycoplasma colonies. If typical mycoplasma colonies appeared within 3 days, species identification was performed using polymerase chain reaction (PCR). However, if no typical mycoplasma colonies grew during the first 3 days of incubation, the culture was continued for a total of 14 days to ensure that the sample was mycoplasma negative (this was subsequently confirmed using PCR). Each broth culture was analyzed using the simplified PCR procedure described below. 2.5. Detection and identification of mycoplasma species by PCR A novel and rapid PCR was used to detect seven mycoplasma species (Mycoplasma bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens, and M. canadense) [19]. A simplified PCR was performed in
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a total reaction volume of 20 mL in an iCycler PCR System (Bio-Rad Laboratories Inc., Richmond, CA, USA). Conditions for the simplified PCR were as follows: initial denaturation at 95 C for 10 minutes followed by 35 cycles of denaturation at 94 C for 30 seconds, annealing at 60 C for 45 seconds, and extension at 72 C for 1 minute. The PCR products were separated by electrophoresis on 1.5% (wt/vol) agarose gels, stained with ethidium bromide, and visualized with an ultraviolet transilluminator. 2.6. Aerobic and anaerobic bacterial culture For a subset of cows (N ¼ 88), uterine swab samples were collected using the cytobrush technique at Weeks 5 and 7. Sterilized swabs were rolled against the cytobrush and then placed in transport medium on the farm and transported to the laboratory. The samples were then plated on a blood agar plate, a chocolate agar plate, and a deoxycholate-hydrogen sulfide-lactose agar plate, and incubated at 35 1 C under aerobic conditions. The samples were also plated on a brain-heart infusion agar plate and incubated at 35 1 C under anaerobic conditions. Colonies present at 24 and 48 hours (aerobic conditions) or at 48 hours (anaerobic conditions) were harvested. When no growth was observed by 48 hours under either aerobic or anaerobic conditions, the samples were plated onto Gifu anaerobic medium semisolid medium and incubated at 35 1 C under aerobic conditions. Bacterial growth was monitored at 24, 48, and 72 hours. If growth was observed on the surface of the medium, the bacteria were extracted from the Gifu anaerobic medium onto plate media and incubated under aerobic conditions. If growth was observed in the deeper part of the media, the extracted bacteria were plated and incubated under anaerobic conditions. Bacterial species were identified by colony morphology, Gram stain, and biochemical characteristics [20]. On the basis of the presence or absence of growth of any bacterial isolates from the uterine samples, cows were considered positive or negative for bacterial infection of the uterus. 2.7. Statistical analysis The incidence of cytologic endometritis and dystocia at the last calving was compared in mycoplasma positive and negative cows using Fisher’s exact probability test. The incidence of dystocia was compared in a subset of cows with uterine bacterial infections using Fisher’s exact probability test. In mycoplasma positive cows, differences between the PMN percentages at Weeks 5 and 7 in cows with and without a history of dystocia at last calving were determined by Student’s t test. Data are presented as mean SEM. Probability values less than 0.05 were considered significant. 3. Results 3.1. Incidence of mycoplasma infection in postpartum cows In total, 418 uterine swabs were collected from 209 cows at Weeks 5 and 7 postpartum. Of these, 31 (7.4%) swabs (15 at
Fig. 1. Molecular diagnosis of Mycoplasma species by polymerase chain reaction, showing the detection of only M. bovigenitalium (450 base pairs [bp]) in Holstein cows. Lane (M), marker of 100 bp; lane (1), uterine isolate of M. bovigenitalium; lane (2), positive control isolate of M. bovigenitalium.
Week 5 and 16 at Week 7) were confirmed positive for M. bovigenitalium (Fig. 1). The mycoplasma positive uterine swabs were collected from 24 cows (eight cows at Week 5, nine at Week 7, and seven at both Weeks 5 and 7). Of the 24 cows infected with M. bovigenitalium, seven (29.2%) had calved during the summer, and the remaining cows had calved from September to December. In addition, 50% of the mycoplasma positive cows were at their first parity. The other six species of mycoplasma were not detected in the uteri of any postpartum cows in this study. 3.2. Incidence of dystocia in mycoplasma positive and negative cows The incidence of dystocia at last calving was higher (P < 0.0001) in mycoplasma positive (29.2%; 7/24) compared with mycoplasma negative (2.2%; 4/185) cows (Table 1). In contrast, the incidence of dystocia at the last calving was not different between cows that cultured positive and negative for other bacteria (P ¼ 0.64; Table 2).
M.E. Ghanem et al. / Theriogenology 79 (2013) 180–185 Table 1 Incidence of dystocia at last calving in relation to M. bovigenitalium infection in Holstein cows. M. bovigenitalium infection
Normal calving
Dystocia
Total
Incidence (%)
P
Negative Positive Total
181 17 198
4 7 11
185 24 209
2.2 29.2 d
<0.0001 d d
3.3. Mycoplasma infection in relation to cytologic endometritis At Week 5, there was no difference in the incidence of cytologic endometritis in mycoplasma positive and mycoplasma negative cows (P ¼ 0.2). However, at Week 7, the incidence of cytologic endometritis was higher (P < 0.05) in mycoplasma positive (50%; 8/16) compared with mycoplasma negative (24.4%; 47/193) cows (Table 3). 3.4. Relationship between dystocia at last calving and cytologic endometritis in mycoplasma positive cows Out of the 17 mycoplasma positive cows that did not experience dystocia at their last calving, five (29.4%) had cytological endometritis. However, five of the seven cows (71.4%) with a history of dystocia at last calving had cytologic endometritis. In mycoplasma positive cows, the percentage of PMN at Week 5 did not differ (P ¼ 0.12) between cows with a history of dystocia at last calving (12.5 9.5) and cows without dystocia (2.6 1.1). However, at Week, 7 cows with a history of dystocia at last calving (12.3 4.7) had a greater PMN score (P < 0.05) compared with cows without dystocia (4.4 1.5). 4. Discussion One of the objectives of this study was to evaluate the incidence of uterine mycoplasma infections. Although it is widely accepted that mycoplasma might cause a variety of clinical symptoms in cattle, the prevalence and reproductive effect of mycoplasma infections in Holstein cows are not well established. In this study, M. bovigenitalium was detected in 7.4% of postpartum Holstein cows. The reported rate of M. bovigenitalium isolated from cervicovaginal mucus samples was 11% in healthy cows [21]. Higher (although not usually significant) isolation rates have been obtained from infertile cows [22], and those with granular vulvitis [12,23]. Moreover, the results of attempts to culture M. bovigenitalium from samples taken from infertile cows
Table 2 Incidence of dystocia at last calving in relation to postpartum uterine bacterial infection in Holstein cows. Postpartum uterine bacterial infection
Normal calving
Dystocia
Total
Incidence (%)
P
Negative Positive Total
35 48 83
1 4 5
36 52 88
2.8 7.7 d
0.64 d d
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have been inconclusive. In one study, only three uteri of 80 abattoir-derived samples from infertile beef cows were positive for mycoplasma: a mixed culture of M. bovigenitalium and M. arginini was recovered from one specimen; M. bovigenitalium in pure culture was recovered from two specimens; and M. bovigenitalium was also isolated from the cotyledons and kidney of one fetus [7]. It is difficult to compare the prevalence of M. bovigenitalium between reports, because of differences in sampling sites and diagnostic methods. However, when comparing the prevalence rate with other studies that examined the cervicovaginal mucus samples, the prevalence of M. bovigenitalium in this study seemed lower than previously reported in healthy animals [13,21], and higher than previously reported in infertile cows [7]. In this study, mycoplasma was obtained from the uterus of postpartum Holstein cows using a cytobrush technique. To the best of our knowledge, this was the first study that has isolated mycoplasma directly from the uterus of living cows. Only a few reports of the isolation of mycoplasma from uteri obtained at slaughter are available [7]. M. bovigenitalium has been recovered from the uterine secretions of cows with breeding problems and endometritis by transrectal massage of the uterine horns [24]. Additionally, M. bovigenitalium was isolated from the cervicovaginal secretions of a cow with pyometra. However, there is considerable doubt that this isolation was truly from the uterus, because the cervical canal was open and the uterus was greatly distended with mucopurulent fluid [10]. There is no available literature on the relationship between dystocia and mycoplasma infection in the uterus of postpartum cows. In the present study, there was a strong positive association between mycoplasma infection and dystocia at last calving. In that regard, nearly 30% of the cows with M. bovigenitalium infection had a history of dystocia. This might be because of the entrance of microbes into the uterus at calving, or to reduced immune function in the genital tracts of cows that have recently experienced dystocia. Interestingly, dystocia was not associated with subsequent postpartum uterine bacterial infection in a subset of cows in the current study. Further investigation is required to elucidate the factors underlying uterine infections, especially in cows with a history of dystocia. After normal parturition the uterus harbors a wide range of microbes; this is mainly attributed to aspiration of environmental contaminants because of the negative uterine pressure created after expulsion of the calf [25]. However, the presence of these microbes does not necessarily indicate infection, and they are usually cleared by effective local and general defense mechanisms in the weeks after calving [26]. However, after an abnormal calving, the microbes might colonize the endometrium and establish infection, leading to puerperal metritis [27]. Prolonged dystocia, uterine inertia, and/or retained fetal membranes can result in the cervix remaining open for a longer interval, thereby providing greater opportunity for bacterial invasion [28,29]. In addition, the likelihood of uterine contamination increases in dystocia cases during the process of examination and assisted delivery of fetus [27]. The bacterial count increases by 12 to 14 hours after birth in bovine dystocia cases [28]. Under normal
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Table 3 Incidence of cytological endometritis at W5 and W7 postpartum in relation to M. bovigenitalium infection in Holstein cows. M. bovigenitalium infection
W5
W7 a
Endometritis
Negative Positive Total
Without
With
138 13 151
56 2 58
Total
194 15 209
Incidence (%)
28.9 13.3
P
0.2
Endometritisa Without
With
146 8 154
47 8 55
Total
Incidence (%)
P
193 16 209
24.4 50.0
0.03
a Diagnostic criteria for cytological endometritis were 6% or more and 4% or more polymorphonuclear neutrophils at Weeks 5 (W5) and 7 (W7), respectively.
circumstances, contaminating organisms are eliminated from the uterus through phagocytosis by migrating leukocytes and through physical expulsion because of uterine contractions. Most of the mycoplasma-positive cows in the present study had a history of dystocia in the summer season. The highest incidence of dystocia was recorded in primipara [30]. There were no statistically significant differences in the rate of occurrence of dystocia in different seasons, but numerically more cases were recorded in the summer, perhaps because of thermal stress [31]. In the present study, some cows were mycoplasma positive at Week 5 and negative at Week 7, suggesting that the uterine immune response eliminated the mycoplasma infection. Other cows were mycoplasma negative at Week 5 and positive at Week 7; this development of infection between Weeks 5 and 7 might be because of a decline in the immune status of the animals. In contrast, it has been previously reported that most cows that are negative for subclinical endometritis at Week 4 postpartum are also negative at Week 8. Similarly, a higher proportion of cows that have subclinical endometritis at Week 4 are also positive at Week 8 postpartum, indicating that subclinical endometritis can persist for a prolonged interval and/or that the same animal can be reinfected on different occasions, although this requires further study [32]. To the best of our knowledge, there are no reports in the literature that describe the persistence of M. bovigenitalium in the bovine uterus. However, mammary infection with M. bovis has been reported to last for varying intervals, from less than 30 days in some cases, to the end of the cow’s lactation in others. An intramammary infection with a high somatic cell count because of M. bovis persisted in some cows for times ranging from 7 weeks to more than 5 months, even after they had returned to apparently normal milk production [33]. These results, like the repeated reisolations of M. bovis over extended intervals from naturally occurring cases of mastitis [34], provided evidence that M. bovis can establish a persistent infection in the bovine mammary gland and that it might cause an extended period of subclinical mastitis after a variable period of clinical M. bovis mastitis [33]. In vitro studies using endometrial cells infected with M. bovigenitalium might provide clues regarding factors associated with the persistence of the organism in the uterine cavity. Little information exists on the relationship between mycoplasma infection and cytological endometritis. One of the novel findings in this study was that the percentage of PMN cells was greater in M. bovigenitalium-positive cows
with a history of dystocia at the last calving than in cows without dystocia. Perhaps M. bovigenitalium aggravated the severity of endometritis when it was associated with dystocia. Furthermore, M. bovigenitalium seemed to be pathogenic to the endometrium and not an opportunistic organism. However, additional investigations in a large population of postpartum cows are required to confirm this. There was no significant association between recent dystocia and uterine infection with bacteria other than mycoplasma. This might be because of sampling technique; we used a cytobrush to collect samples from the uterine body for bacterial culture. Although previous studies have shown that the sensitivity of cytobrush cytology for diagnosing subclinical endometritis is high [18], it is possible that some pathogens cannot be isolated from samples collected in this manner. It has been previously reported that, even in samples with cytologic evidence of inflammation and phagocytized bacteria, culture of uterine biopsies taken at the same time might be negative for bacteria. Consequently, even bacteriological investigation of uterine biopsies did not detect all cases of infection [35]. In addition, the cytobrush might touch only a small area of the endometrium and focal infections might be missed [36]. In this study, we did not examine or sample the animals after Week 7 postpartum. Future studies should focus on the follow-up of the infected cows to evaluate the pathogenicity of mycoplasma, the ability of cows to self-cure the infection, and the subsequent reproductive performance of infected animals. In addition, the significance of M. bovigenitalium infection in other places in the body besides the uterus should be evaluated. 4.1. Conclusions Out of seven species of mycoplasma, only M. bovigenitalium was detected in the uterus of postpartum cows. M. bovigenitalium infection has an incidence of 7.4% and might be associated with dystocia at last calving and with cytological endometritis in postpartum dairy cows. Acknowledgments M. E. Ghanem is supported by a Japan Society for the Promotion of Science (JSPS) fellowship. The authors thank Miyarisan Pharmaceutical Co. Ltd., for providing mycoplasma culture broth, and the members of the Food
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