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Ovarian hydrobursitis in female camels (Camelus dromedarius): The role of Chlamydophila abortus and a trial for medical treatment
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Theriogenology 77 (2012) 1754–1758 www.theriojournal.com
Ovarian hydrobursitis in female camels (Camelus dromedarius): The role of Chlamydophila abortus and a trial for medical treatment A. Alia,*, F.A. Al-Sobayila, K.M. Hassaneinb, A. Al-Hawasa a
Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim University, 51452 Buraydah, Qassim, Saudi Arabia b Department of Pathology, College of Medicine, Qassim University, 51452 Buraydah, Qassim, Saudi Arabia Received 13 October 2011; received in revised form 9 December 2011; accepted 9 December 2011
Abstract The occurrence of Chlamydophila abortus in female camels affected with ovarian hydrobursitis and a trial for medical treatment were studied. A total of 111 cases were included in two experiments. In Experiment 1, sera from 51 affected cases were tested for C. abortus antibody using enzyme-linked immunosorbent assay (ELISA). In Experiment 2, 60 female camels affected with bilateral ovarian hydrobursitis were divided into treated and control groups (n ⫽ 30 each). Based on the bursal diameter, females of both groups were subdivided into those having small (⬍ 5 cm), medium (5–7 cm) or large (⬎ 7 cm) bursae. Treated group received 20 mg/kg body weight oxytetracycline intramuscular, 4% lotagen intrauterine, and 500 g cloprostenol intramuscular. Controls did not receive any treatment. All females were observed for 90 days non-return rate (NRR) and calving rate (CR). Antibodies against C. abortus were observed in 44/51 (86.3%) of the affected females. The 90 days NRR of the treated and control groups were 13/30 (43.3%) and 0/30 (0.0%), respectively, (P ⫽ 0.001), while the CR were 10/30 (33.3%) and 0/30 (0.0%), respectively, (P ⫽ 0.01). Based on bursal size, the 90 days NRR were 11/15 (73.3%), 2/7 (28.6%) and 0/8 (0.0%) for treated females having small, medium and large bursa, while the CR were 9/15 (60%), 1/7 (14.3%), and 0/8 (0.0%), respectively, (P ⫽ 0.01). In conclusion, it seems that C. abortus may be responsible for the spreading of the ovarian hydrobursitis syndrome in dromedaries. Small sized bursa could be medically treated. © 2012 Elsevier Inc. All rights reserved. Keywords: Female camels; Infertility; Ovarian hydrobursitis; Chlamydophila abortus; Treatment
1. Introduction Ovarian hydrobursitis is a serious long-standing reproductive syndrome in dromedary camels characterized by fluid accumulation and encapsulation of the ovary [1–3]. It is manifested by early embryonic death, abortion, repeat breeding, and refuse mating. Ovarian hydrobursitis represented 30 to 40% of infer-
* Corresponding author. Tel.: 0020882295574; fax: 0020882333938. E-mail address: [email protected] (A. Ali). 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.12.017
tile cases in dromedary camels and associated with purulent endometritis, adhesions of the uterus and vagina, closed cervix, pyometra, and enlargement of the fallopian tube [2,3]. Several trials have been proceeded to clarify the etiopathogenesis of this syndrome. It has been suggested because of inflammatory reaction that might be caused by a specific pathogen [2,3]. The epidemiologic importance of Aeromonas hydrophila. Trypansoma evansii, and hydatid cyst have been discussed as potential causative pathogens [2– 4]. However, chla-
A. Ali et al. / Theriogenology 77 (2012) 1754–1758
mydia infection has not been considered in any of these studies. Chlamydia is a common etiologic factor of salpingitis and cystic ovarian bursa in mice [5,6]. It has been cultured from the bursa and elsewhere in the genital tract of experimentally infected koala (Phascolarctos cinereus) [7]. In women, Chlamydia is a common sexually transmitted infection that can spread into the fallopian tubes and causes tubal block at the very ends [8 –10]. In llama and alpaca, leptospirosis, toxoplasmosis and chlamydiosis have been diagnosed as the major causes of abortion, while in dromedary; brucellosis and trypanosomiasis represented the major causes of infectious abortion in the Middle East and Africa [11]. Surgical ablation of the affected bursa in unilaterally affected cases has been recommended as conception rates after these trials were greatly acceptable [1–3]. However, surgical ablation in bilaterally affected cases could not be considered and therefore alternative approach was intended. The aim of this study was to investigate the role of Chlamydophila abortus as a causative agent for the ovarian hydrobursitis syndrome in dromedary camels. Medical treatment of cases with bilateral infection was also investigated. 2. Materials and methods 2.1. Experiment 1: detection of C. abortus antibodies in cases with ovarian hydrobursitis Based on transrectal palpation (difficult in retraction of the genital tract and/or in catching the ovary) and ultrasound examination (presence of a hypoechogenic well-demarcated sac encapsulating the ovary), 51 infertile female dromedary camels (age from 5 to 13 yrs) were diagnosed as having ovarian hydrobursitis syndrome (24 unilateral and 27 bilateral). The examinations were performed at the Veterinary Teaching Hospital of Qassim University, Saudi Arabia, during the breeding season (September 2009 to March 2010) for cases attending for different infertility problems. Transrectal ultrasound examination was done using Aloka SSD-500 machine equipped with 5 or 7 MHz lineararray transducers (Aloka Co., Ltd, Tokyo, Japan). Females were also examined for general health condition and for body condition score using a scale from 1 to 5 [12]. Camels were nullipara (n ⫽ 12) and multipara (n ⫽ 39). On admission, jugular blood samples (10 mL) were taken from all animals (n ⫽ 51) into plane tubes. Serum was separated by centrifugation for 15 min at1200 xg
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and was immediately frozen at ⫺20 °C for consequent analyses. Enzyme-linked immunosorbent assay (ELISA) was used to detect IgG antibodies against C. abortus (kits provided by IDEXX Switzeland AG, Liebelfeld-Bern, Switherland). Microtiter plates were supplied precoated with inactivated antigen. Samples to be tested were incubated in the wells of the plates. Any antibody directed against C. abortus bind to the antigen in the wells and formed an antigen/antibody complex on the plate well surface. Unbound material was removed from the wells by washing. A peroxidase labeled antiruminant IgG conjugate was added, which bind to the ruminant antibodies complexed with the C. abortus antigen. Unbound conjugate was removed by washing, and the substrate was added to the wells. The degree of color that develops was directly proportional to the amount of antibody specific for C. abortus present in the sample. The diagnostic relevance of the result was obtained by comparing the optical density of the sample well with the optical density from the positive control. 2.2. Experiment 2: treatment regime for cases with bilateral ovarian hydrobursitis Sixty dromedary camels with bilateral ovarian hydrobursitis were randomly categorized into treated (n ⫽ 30) and non-treated (n ⫽ 30) groups. According to the maximum diameter of the affected bursa, animals in treated group were subdivided into those having small (⬍ 5 cm, n ⫽ 15), medium (5–7 cm, n ⫽ 7), and large (⬎ 7 cm, n ⫽ 8) bursa. Similarly, non-treated group were subdivided into those with small (n ⫽ 13), medium (n ⫽ 10), and large, (n ⫽ 7) bursa (Fig. 1). Female camels in treated group received 20 mg/kg body weight oxytetracycline (Terramycin, Pfizer, Ltda, Animal health division, Sao Paulo, Brazil) administrated intramuscular for 7 days; 100 mL 4% lotagen solution (metacresolsulphonic acid and formaldehyde, Schering-Plough Animal Health, Segre-France) administrated intrauterine once at day of attendance; and 500 g cloprostenol (PGF2␣ analogue, Estrumate, Schering-Plough, Morris Ave, Summit, NJ) given intramuscular at infusion time. The non-treated group did not receive any medicaments. The females were mated during the first estrus just after treatment completed. 2.3. Fertility Treated female camels and controls were then observed for the 90 days non-return rate (NRR) and calving rate (CR). This study followed the guidelines ad-
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Fig. 1. Ovarian hydrobursitis in female camels of small (A, 2.2 cm in diameter), medium (B, 5.7 cm in diameter) and large (C, 7.2 cm in diameter) sizes; 1: ovary, 2: bursa.
opted by the Animal Welfare and Scientific and Research Deanship, Qassim University. 2.4. Statistical analysis The differences in the percentages of the 90 NRR and CR between treated and non-treated animals were evaluated by the 2 test. While, these differences among small, medium, and large sized bursae were analyzed by Kruskal-Wallis test. The level of significance was tested at P ⬍ 0.05. A statistical program (SPSS, [13]) was used to perform the statistical analysis. 3. Results On admission, none of female camels with ovarian hydrobursitis showed fever or reduced appetite. Heart and respiratory rates as well as ruminal movements were within the normal values. Animal body condition score ranged from 2.5 to 3.5. Females had histories of reproductive disorders [early embryonic death (n ⫽ 63/111, 56.8%); abortion (n ⫽ 19/111, 17.1%); repeat
breading (n ⫽ 17/111, 15.3%); refuse mating (13/111, 11.7%)] for a period ranging from 6 mo to 2 yrs prior to admission. On 18 occasions, 2 to 4 affected females were presented from the same herd. Antibodies against C. abortus were observed in 44/51 (86.3%) of the female camels affected with ovarian hydrobursitis, one case (2%) was suspected and the others (11.7%) were negative. The incidence of positive cases was approximately equal in unilaterally (20/24, 83.3%) and bilaterally (24/27, 88.9%) affected animals as well as in nullipara (10/12, 83.3%) and multipara (34/39, 87.2%). The 90 days NRR and CR were significantly influenced by the treatment (P ⫽ 0.001) (Table 1) and size of the affected bursa (P ⫽ 0.01) (Table 2). 4. Discussion This is the first report associates titers of serum immunoglobulin G (IgG) against Chlamydophila abortus with the occurrence of the ovarian hydrobursitis syndrome in dromedary camels. C. abortus is a species
Table 1 The 90 d NRR and CR in treateda (n ⫽ 30) and non-treated (n ⫽ 30) dromedaries affected with ovarian hydrobursitis.
Table 2 Effect of bursal size on the efficacy of treatment of dromedaries with ovarian hydrobursitis (n ⫽ 30).
Regime
90 Days NRR
CR
Size of bursa
90 Days NRR
CR
Treated Non-treated
13/30a (43.3%) 0/30b (0.0%)
10/30a (33.3%) 0/30b (0.0%)
Small (⬍3 cm) Medium (3–5 cm) Large (⬎5 mm)
11/15a (73.3%) 2/7b (28.6%) 0/8b (0.0%)
9/15a (60%) 1/7b (14.3%) 0/8b (0.0%)
a a,b
Treatment consisted of oxytetracyclin ⫹ lotagen ⫹cloprostenol. Values with different letters in the same column differ significantly.
a,b
Values with different letters in the same column differ significantly.
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in Chlamydiae that causes abortion and fetal death in mammals, including humans. The organism has been previously classified as Chlamydia psittaci along with all Chlamydiae except C. trachomatis. This Gram-negative bacterium is an obligate intracellular pathogen of eukaryotic cells. Since C. abortus is a pathogen, this organism depends on its host for its proper nutrients and energy [14]. Based on this Study C. Abortus may be playing a major role in the etiopathogensis of the ovarian hydrobursitis syndrome in dromedary camels. C. abortus had histories of many reproductive problems in livestock worldwide. A study done in 2006, examined the abortion rate of a group of sheep and goats in Hungary. The study took place over seven and a half year period. It has been found that 63% of all the abortions were due to C. abortus [15]. Endometritis in cattle has been experimentally induced by C. abortus [16]. Moreover, C. abortus has been used to infect bovine oviduct cells. After infection, inclusion bodies and vacuoles formed in the cells; the cells appeared to be damaged and the microvilli were congregated and stuck together, this causes infertility within the animals [17]. Camels which have been bred for the purpose of racing have been tested serologically and 24% of them were positive for chlamydiosis [18]. This study used ELISA to detect chlamydia infection. The most common test to detect chlamydia infection in women involves taking a swab from the cervix during a speculum examination and testing it for chlamydia DNA [19]. Blood tests have been also used to detect the chlamydia antibodies made by the body when exposed to chlamydia infection. These blood tests have been found fairly predictive for finding women with tubal damage [20]. Polymerase chain reaction (PCR), transcription mediated amplification (TMA), and the DNA strand displacement amplification (SDA) now are the mainstays [21]. The swab test, however, can miss an infection that has moved up into the uterus or tubes and is no longer in the cervix, especially in chronic infection [22]. In sheep, definitive diagnosis has been observed by identification of C. abortus by ELISA [23]. Additionally, ELISA is comparatively of lower cost and ease to performance. In this study, more than one female were submitted at the same time from one herd, supporting contribution of a specific infectious agent. Male camels may be incriminated in transmitting the chlamydial infection. Chlamydiae infect male genital organs of ruminants [24,25] and cause prostatitis and epididymitis in men [26].
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According to the current study, small sized ovarian hydrobursitis could be treated by a combination of oxytertacycline, intrauterine lotagen irrigation and prostaglandin administration. Chlamydiosis is best responds to tetracycline in humans, sheep and goats. In humans, up to 95% of people with chlamydiosis have been cured after one course of antibiotics. Azithromycin, doxycycline, tetracycline, or erythromycin have been recommended [27]. Chlamydophila abortus (Chlamydia psittaci serotype 1) is the cause of enzootic abortion of ewes (EAE), which is characterized by late term abortions, stillbirths, and weak lambs. Control consists of isolating all affected ewes and lambs and treating in-contact ewes with long acting oxytetracycline or oral tetracycline [28,29]. Prostaglandin causes luteolysis of a responsive corpus luteum resulting in decreased progesterone level and subsequent estrus, with increased estrogen level and myometrial contractions. These events are all reasonably favorable for clearance of uterine infections [30]. In addition, prostaglandin has direct effects on the uterus, and can resolve uterine infections in cows without corpus luteum [31]. Lotagen solution attacks the cell wall of bacteria directly, coagulates mucus and debris and supports the processes of granulation and epithelization [32]. Medium and large sized bursae did not respond to the current treatment regime. Authors think that the excessive fluid accumulation, distension of the fallopian tube and/or development of ovarian adhesions might be the causes of this conception failure in camels of this study. Since the occurrence of this syndrome is very high among the dromedaries, it may be feasible to converse other possibilities of preventing this syndrome like vaccination of animals. Protective immunity was shown to be induced in sheep with a vaccine consisting of C. abortus grown in fertile hens’ eggs and subsequently inactivated and incorporated with an oily adjuvant [33]. In parts of Europe, a modified live vaccine is available for use to control C. abortus in sheep [29]. In conclusion, C. abortus may be responsible for the spreading of the ovarian hydrobursitis syndrome in dromedaries. Small sized ovarian hydrobursitis (⬍5 cm in diameter) could be medically treated.
Acknowledgments This study was supported by the Deanship for Scientific Research, Qassim University, Saudi Arabia.
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References [1] Tibary A, Anouassi A. Retrospective study on an unusual form of ovario-bursal pathology in the camel (Camelus dromedarius). Theriogenology 2001;56:415–24. [2] Ali A, Al-Sobayil FA, Tharwat M, Hassanein KM. Ovarian hydrobursitis in female camels (Camelus dromedaries): biochemical, bacterial and protozoal evaluation. Theriogenology 2011;75:734 – 41. [3] Ali A, Mehana EE, Ahmed AF, El-Tookhy O, Al-Sobayil A, Al-Hawas A. Ovarian hydrobursitis in female camels (Camelus dromedarius): clinical findings, histopathology and fertility after unilateral surgical ablation. Theriogenology 2011;76:492–9. [4] Al-Eknah MM, Ali AMA. Infundibular cyst jeopardize reproduction in female camels (Camelus dromedarius). Emir J Agri Sci 2001;13:52–56. [5] Swenson CE, Donegan E, Schachter J. Chlamydia trachomatisinduced salpingitis in mice. J Infect Dis 1983;148:1101–7. [6] Khamesipour Pal S, Peterson EM, de la Maza LM, de la Maza LM. Induction of infertility by the Chlamydia trachomatis mouse pneumonitis biovar in strains of mice that differ in their response to the 60 kDa heat shock protein. J Reprod Fertil 1994;101:287–94. [7] Obendorf DL, Handasyde KA. Pathology of chlamydial infection in the reproductive tract of the female koala. In: Lee AK, Handasyde KA, Sanson GD, editors. Biology of the koala. Sydney, Australia: Surrey Beatty and Sons; 1990. [8] LaVerda D, Albanese LN, Ruther PE, Morrison SG, Morrison RP, Ault KA, et al. Seroreactivity to Chlamydia trachomatis Hsp10 correlates with severity of human genital tract disease. Infect Immun 2000;68:303–9. [9] Higgins DP, Hemsley S, Canfield PJ. Association of uterine and salpingeal fibrosis with chlamydial hsp60 and hsp10 antigenspecific antibodies in chlamydia-infected koalas. Clin Diagn Lab Immunol 2005;12:632–9. [10] Faber MT, Nielsen A, Nygård M, Sparén P, Tryggvadottir L, Hansen BT, et al. Genital chlamydia, genital herpes, Trichomonas vaginalis and gonorrhea prevalence, and risk factors among nearly 70,000 randomly selected women in 4 Nordic countries. Sex Transm Dis 2011;38:727–34. [11] Tibary A, Fite C, Anouassi A, Sghiri A. Infectious causes of reproductive loss in camelids. Theriogenology 2006;66: 633– 47. [12] Sghiri A, Driancourt MA. Seasonal effects on fertility and ovarian follicular growth and maturation in camels (Camelus dromedarius). Anim Reprod Sci 1999;55:223–37. [13] SPSS. Statistical Package for Social Sciences. Chicago, IL, USA: SPSS, Inc. Copyright for Windows, version 18.0. [14] Everett KD, Bush RM, Andersen AA. Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. Int J Syst Bacteriol 1999;49: 415– 40. [15] Szeredi L, Jánosi S, Tenk M, Tekes L, Bozsó M, Deim Z, et al. Epidemiological and pathological study on the causes of abortion in sheep and goats in Hungary (1998-2005). Acta Vet Hung 2006;54:503–15.
[16] Wittenbrink MM, Schoon HA, Schoon D, Mansfeld R, Bisping W. Endometritis in cattle experimentally induced by Chlamydia psittaci. Zentralbl Veterinarmed B 1993;40:437–50. [17] Appino S, Pregel P, Manuali E, Vincenti L, Rota A, Carnieletto P, et al. Infection of bovine oviduct cell cultures with Chlamydophila abortus. Anim Reprod Sci 2007;98:350 – 6. [18] Wernery U, Wernery R. [Seroepidemiologic studies of the detection of antibodies to Brucella, Chlamydia, Leptospira, BVD/MD virus, IBR/IPV virus and enzootic bovine leukosis virus (EBL) in dromedary mares (Camelus dromedarius)]. Dtsch Tierarztl Wochenschr 1990;97:134 –5. [19] Khan ER, Hossain MA, Paul SK, Mahmud MC, Rahman MM, Alam MA, et al. Molecular diagnosis of genital Chlamydia trachomatis infection by polymerase chain reaction. Mymensingh Med J 2011;20:362–5. [20] Baud D, Regan L, Greub G. Comparison of five commercial serological tests for the detection of anti-Chlamydia trachomatis antibodies. Eur J Clin Microbiol Infect Dis 2010;29:669 –75. [21] Khan ER, Hossain MA, Paul SK, Mahmud MC, Rahman MM, Alam MA, et al. Molecular diagnosis of genital Chlamydia trachomatis infection by polymerase chain reaction Mymensingh Med J 2011;20:362–5. [22] Chernesky MA. The laboratory diagnosis of Chlamydia trachomatis infections. Can J Infect Dis Med Microbiol 2005;16: 39 – 44. [23] Donn A, Jones GE, Ruiu A, Ladu M, Machell J, Stancanelli A. Serological diagnosis of chlamydial abortion in sheep and goats: comparison of the complement fixation test and an enzymelinked immunosorbent assay employing solubilised proteins as antigen. Vet Microbiol 1997;59:27–36. [24] Teankum K, Pospischil A, Janett F, Brugnera E, Hoelzle LE, Hoelzle K, et al. Prevalence of chlamydiae in semen and genital tracts of bulls, rams and bucks. Theriogenology 2007;67: 303–10. [25] Kauffold J, Henning K, Bachmann R, Hotzel H, Melzer F. The prevalence of chlamydiae of bulls from six bull studs in Germany. Anim Reprod Sci 2007;102:111–21. [26] Wagenlehner FM, Naber KG, Weidner W. Chlamydial infections and prostatitis in men. BJU Int 2006;97:687–90. [27] West SK, Munoz B, Mkocha H, Gaydos CA, Quinn TC. Number of years of annual mass treatment with azithromycin needed to control trachoma in hyper-endemic communities in Tanzania. J Infect Dis 2011;204:268 –73. [28] Greig A, Linklater KA. Field studies on the efficacy of a long acting preparation of oxytetracycline in controlling outbreaks of enzootic abortion of sheep. Vet Rec 1985;117:627– 8. [29] Kahn CM and Scott L, editors. The Merck Veterinary Manual. Wiley, 10th Edition 2010. [30] Weems CW, Weems YS, Randel RD. Prostaglandins and reproduction in female farm animals. Vet J 2006;171:206 –28. [31] LeBlanc SJ, Duffield TF, Leslie KE, Bateman KG, Keefe GP, Walton JS, et al. Defining and diagnosing postpartum clinical endometritis and its impact on reproductive performance in dairy cows. J Dairy Sci 2002;85:2223–36. [32] Schnyder D, Küpfer U, Zwahlen R, Schnyder D, Küpfer U, Zwahlen R. [Changes in the endometrium of the cow after intrauterine administration of different drugs]. Schweiz Arch Tierheilkd 1990;132:353– 64. [33] McEwen AD, Foggie A. Enzootic abortion of ewes; comparative studies of different vaccines. Vet Rec 1954;66:393–7.
Theriogenology 77 (2012) 1754–1758 www.theriojournal.com
Ovarian hydrobursitis in female camels (Camelus dromedarius): The role of Chlamydophila abortus and a trial for medical treatment A. Alia,*, F.A. Al-Sobayila, K.M. Hassaneinb, A. Al-Hawasa a
Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim University, 51452 Buraydah, Qassim, Saudi Arabia b Department of Pathology, College of Medicine, Qassim University, 51452 Buraydah, Qassim, Saudi Arabia Received 13 October 2011; received in revised form 9 December 2011; accepted 9 December 2011
Abstract The occurrence of Chlamydophila abortus in female camels affected with ovarian hydrobursitis and a trial for medical treatment were studied. A total of 111 cases were included in two experiments. In Experiment 1, sera from 51 affected cases were tested for C. abortus antibody using enzyme-linked immunosorbent assay (ELISA). In Experiment 2, 60 female camels affected with bilateral ovarian hydrobursitis were divided into treated and control groups (n ⫽ 30 each). Based on the bursal diameter, females of both groups were subdivided into those having small (⬍ 5 cm), medium (5–7 cm) or large (⬎ 7 cm) bursae. Treated group received 20 mg/kg body weight oxytetracycline intramuscular, 4% lotagen intrauterine, and 500 g cloprostenol intramuscular. Controls did not receive any treatment. All females were observed for 90 days non-return rate (NRR) and calving rate (CR). Antibodies against C. abortus were observed in 44/51 (86.3%) of the affected females. The 90 days NRR of the treated and control groups were 13/30 (43.3%) and 0/30 (0.0%), respectively, (P ⫽ 0.001), while the CR were 10/30 (33.3%) and 0/30 (0.0%), respectively, (P ⫽ 0.01). Based on bursal size, the 90 days NRR were 11/15 (73.3%), 2/7 (28.6%) and 0/8 (0.0%) for treated females having small, medium and large bursa, while the CR were 9/15 (60%), 1/7 (14.3%), and 0/8 (0.0%), respectively, (P ⫽ 0.01). In conclusion, it seems that C. abortus may be responsible for the spreading of the ovarian hydrobursitis syndrome in dromedaries. Small sized bursa could be medically treated. © 2012 Elsevier Inc. All rights reserved. Keywords: Female camels; Infertility; Ovarian hydrobursitis; Chlamydophila abortus; Treatment
1. Introduction Ovarian hydrobursitis is a serious long-standing reproductive syndrome in dromedary camels characterized by fluid accumulation and encapsulation of the ovary [1–3]. It is manifested by early embryonic death, abortion, repeat breeding, and refuse mating. Ovarian hydrobursitis represented 30 to 40% of infer-
* Corresponding author. Tel.: 0020882295574; fax: 0020882333938. E-mail address: [email protected] (A. Ali). 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.12.017
tile cases in dromedary camels and associated with purulent endometritis, adhesions of the uterus and vagina, closed cervix, pyometra, and enlargement of the fallopian tube [2,3]. Several trials have been proceeded to clarify the etiopathogenesis of this syndrome. It has been suggested because of inflammatory reaction that might be caused by a specific pathogen [2,3]. The epidemiologic importance of Aeromonas hydrophila. Trypansoma evansii, and hydatid cyst have been discussed as potential causative pathogens [2– 4]. However, chla-
A. Ali et al. / Theriogenology 77 (2012) 1754–1758
mydia infection has not been considered in any of these studies. Chlamydia is a common etiologic factor of salpingitis and cystic ovarian bursa in mice [5,6]. It has been cultured from the bursa and elsewhere in the genital tract of experimentally infected koala (Phascolarctos cinereus) [7]. In women, Chlamydia is a common sexually transmitted infection that can spread into the fallopian tubes and causes tubal block at the very ends [8 –10]. In llama and alpaca, leptospirosis, toxoplasmosis and chlamydiosis have been diagnosed as the major causes of abortion, while in dromedary; brucellosis and trypanosomiasis represented the major causes of infectious abortion in the Middle East and Africa [11]. Surgical ablation of the affected bursa in unilaterally affected cases has been recommended as conception rates after these trials were greatly acceptable [1–3]. However, surgical ablation in bilaterally affected cases could not be considered and therefore alternative approach was intended. The aim of this study was to investigate the role of Chlamydophila abortus as a causative agent for the ovarian hydrobursitis syndrome in dromedary camels. Medical treatment of cases with bilateral infection was also investigated. 2. Materials and methods 2.1. Experiment 1: detection of C. abortus antibodies in cases with ovarian hydrobursitis Based on transrectal palpation (difficult in retraction of the genital tract and/or in catching the ovary) and ultrasound examination (presence of a hypoechogenic well-demarcated sac encapsulating the ovary), 51 infertile female dromedary camels (age from 5 to 13 yrs) were diagnosed as having ovarian hydrobursitis syndrome (24 unilateral and 27 bilateral). The examinations were performed at the Veterinary Teaching Hospital of Qassim University, Saudi Arabia, during the breeding season (September 2009 to March 2010) for cases attending for different infertility problems. Transrectal ultrasound examination was done using Aloka SSD-500 machine equipped with 5 or 7 MHz lineararray transducers (Aloka Co., Ltd, Tokyo, Japan). Females were also examined for general health condition and for body condition score using a scale from 1 to 5 [12]. Camels were nullipara (n ⫽ 12) and multipara (n ⫽ 39). On admission, jugular blood samples (10 mL) were taken from all animals (n ⫽ 51) into plane tubes. Serum was separated by centrifugation for 15 min at1200 xg
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and was immediately frozen at ⫺20 °C for consequent analyses. Enzyme-linked immunosorbent assay (ELISA) was used to detect IgG antibodies against C. abortus (kits provided by IDEXX Switzeland AG, Liebelfeld-Bern, Switherland). Microtiter plates were supplied precoated with inactivated antigen. Samples to be tested were incubated in the wells of the plates. Any antibody directed against C. abortus bind to the antigen in the wells and formed an antigen/antibody complex on the plate well surface. Unbound material was removed from the wells by washing. A peroxidase labeled antiruminant IgG conjugate was added, which bind to the ruminant antibodies complexed with the C. abortus antigen. Unbound conjugate was removed by washing, and the substrate was added to the wells. The degree of color that develops was directly proportional to the amount of antibody specific for C. abortus present in the sample. The diagnostic relevance of the result was obtained by comparing the optical density of the sample well with the optical density from the positive control. 2.2. Experiment 2: treatment regime for cases with bilateral ovarian hydrobursitis Sixty dromedary camels with bilateral ovarian hydrobursitis were randomly categorized into treated (n ⫽ 30) and non-treated (n ⫽ 30) groups. According to the maximum diameter of the affected bursa, animals in treated group were subdivided into those having small (⬍ 5 cm, n ⫽ 15), medium (5–7 cm, n ⫽ 7), and large (⬎ 7 cm, n ⫽ 8) bursa. Similarly, non-treated group were subdivided into those with small (n ⫽ 13), medium (n ⫽ 10), and large, (n ⫽ 7) bursa (Fig. 1). Female camels in treated group received 20 mg/kg body weight oxytetracycline (Terramycin, Pfizer, Ltda, Animal health division, Sao Paulo, Brazil) administrated intramuscular for 7 days; 100 mL 4% lotagen solution (metacresolsulphonic acid and formaldehyde, Schering-Plough Animal Health, Segre-France) administrated intrauterine once at day of attendance; and 500 g cloprostenol (PGF2␣ analogue, Estrumate, Schering-Plough, Morris Ave, Summit, NJ) given intramuscular at infusion time. The non-treated group did not receive any medicaments. The females were mated during the first estrus just after treatment completed. 2.3. Fertility Treated female camels and controls were then observed for the 90 days non-return rate (NRR) and calving rate (CR). This study followed the guidelines ad-
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Fig. 1. Ovarian hydrobursitis in female camels of small (A, 2.2 cm in diameter), medium (B, 5.7 cm in diameter) and large (C, 7.2 cm in diameter) sizes; 1: ovary, 2: bursa.
opted by the Animal Welfare and Scientific and Research Deanship, Qassim University. 2.4. Statistical analysis The differences in the percentages of the 90 NRR and CR between treated and non-treated animals were evaluated by the 2 test. While, these differences among small, medium, and large sized bursae were analyzed by Kruskal-Wallis test. The level of significance was tested at P ⬍ 0.05. A statistical program (SPSS, [13]) was used to perform the statistical analysis. 3. Results On admission, none of female camels with ovarian hydrobursitis showed fever or reduced appetite. Heart and respiratory rates as well as ruminal movements were within the normal values. Animal body condition score ranged from 2.5 to 3.5. Females had histories of reproductive disorders [early embryonic death (n ⫽ 63/111, 56.8%); abortion (n ⫽ 19/111, 17.1%); repeat
breading (n ⫽ 17/111, 15.3%); refuse mating (13/111, 11.7%)] for a period ranging from 6 mo to 2 yrs prior to admission. On 18 occasions, 2 to 4 affected females were presented from the same herd. Antibodies against C. abortus were observed in 44/51 (86.3%) of the female camels affected with ovarian hydrobursitis, one case (2%) was suspected and the others (11.7%) were negative. The incidence of positive cases was approximately equal in unilaterally (20/24, 83.3%) and bilaterally (24/27, 88.9%) affected animals as well as in nullipara (10/12, 83.3%) and multipara (34/39, 87.2%). The 90 days NRR and CR were significantly influenced by the treatment (P ⫽ 0.001) (Table 1) and size of the affected bursa (P ⫽ 0.01) (Table 2). 4. Discussion This is the first report associates titers of serum immunoglobulin G (IgG) against Chlamydophila abortus with the occurrence of the ovarian hydrobursitis syndrome in dromedary camels. C. abortus is a species
Table 1 The 90 d NRR and CR in treateda (n ⫽ 30) and non-treated (n ⫽ 30) dromedaries affected with ovarian hydrobursitis.
Table 2 Effect of bursal size on the efficacy of treatment of dromedaries with ovarian hydrobursitis (n ⫽ 30).
Regime
90 Days NRR
CR
Size of bursa
90 Days NRR
CR
Treated Non-treated
13/30a (43.3%) 0/30b (0.0%)
10/30a (33.3%) 0/30b (0.0%)
Small (⬍3 cm) Medium (3–5 cm) Large (⬎5 mm)
11/15a (73.3%) 2/7b (28.6%) 0/8b (0.0%)
9/15a (60%) 1/7b (14.3%) 0/8b (0.0%)
a a,b
Treatment consisted of oxytetracyclin ⫹ lotagen ⫹cloprostenol. Values with different letters in the same column differ significantly.
a,b
Values with different letters in the same column differ significantly.
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in Chlamydiae that causes abortion and fetal death in mammals, including humans. The organism has been previously classified as Chlamydia psittaci along with all Chlamydiae except C. trachomatis. This Gram-negative bacterium is an obligate intracellular pathogen of eukaryotic cells. Since C. abortus is a pathogen, this organism depends on its host for its proper nutrients and energy [14]. Based on this Study C. Abortus may be playing a major role in the etiopathogensis of the ovarian hydrobursitis syndrome in dromedary camels. C. abortus had histories of many reproductive problems in livestock worldwide. A study done in 2006, examined the abortion rate of a group of sheep and goats in Hungary. The study took place over seven and a half year period. It has been found that 63% of all the abortions were due to C. abortus [15]. Endometritis in cattle has been experimentally induced by C. abortus [16]. Moreover, C. abortus has been used to infect bovine oviduct cells. After infection, inclusion bodies and vacuoles formed in the cells; the cells appeared to be damaged and the microvilli were congregated and stuck together, this causes infertility within the animals [17]. Camels which have been bred for the purpose of racing have been tested serologically and 24% of them were positive for chlamydiosis [18]. This study used ELISA to detect chlamydia infection. The most common test to detect chlamydia infection in women involves taking a swab from the cervix during a speculum examination and testing it for chlamydia DNA [19]. Blood tests have been also used to detect the chlamydia antibodies made by the body when exposed to chlamydia infection. These blood tests have been found fairly predictive for finding women with tubal damage [20]. Polymerase chain reaction (PCR), transcription mediated amplification (TMA), and the DNA strand displacement amplification (SDA) now are the mainstays [21]. The swab test, however, can miss an infection that has moved up into the uterus or tubes and is no longer in the cervix, especially in chronic infection [22]. In sheep, definitive diagnosis has been observed by identification of C. abortus by ELISA [23]. Additionally, ELISA is comparatively of lower cost and ease to performance. In this study, more than one female were submitted at the same time from one herd, supporting contribution of a specific infectious agent. Male camels may be incriminated in transmitting the chlamydial infection. Chlamydiae infect male genital organs of ruminants [24,25] and cause prostatitis and epididymitis in men [26].
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According to the current study, small sized ovarian hydrobursitis could be treated by a combination of oxytertacycline, intrauterine lotagen irrigation and prostaglandin administration. Chlamydiosis is best responds to tetracycline in humans, sheep and goats. In humans, up to 95% of people with chlamydiosis have been cured after one course of antibiotics. Azithromycin, doxycycline, tetracycline, or erythromycin have been recommended [27]. Chlamydophila abortus (Chlamydia psittaci serotype 1) is the cause of enzootic abortion of ewes (EAE), which is characterized by late term abortions, stillbirths, and weak lambs. Control consists of isolating all affected ewes and lambs and treating in-contact ewes with long acting oxytetracycline or oral tetracycline [28,29]. Prostaglandin causes luteolysis of a responsive corpus luteum resulting in decreased progesterone level and subsequent estrus, with increased estrogen level and myometrial contractions. These events are all reasonably favorable for clearance of uterine infections [30]. In addition, prostaglandin has direct effects on the uterus, and can resolve uterine infections in cows without corpus luteum [31]. Lotagen solution attacks the cell wall of bacteria directly, coagulates mucus and debris and supports the processes of granulation and epithelization [32]. Medium and large sized bursae did not respond to the current treatment regime. Authors think that the excessive fluid accumulation, distension of the fallopian tube and/or development of ovarian adhesions might be the causes of this conception failure in camels of this study. Since the occurrence of this syndrome is very high among the dromedaries, it may be feasible to converse other possibilities of preventing this syndrome like vaccination of animals. Protective immunity was shown to be induced in sheep with a vaccine consisting of C. abortus grown in fertile hens’ eggs and subsequently inactivated and incorporated with an oily adjuvant [33]. In parts of Europe, a modified live vaccine is available for use to control C. abortus in sheep [29]. In conclusion, C. abortus may be responsible for the spreading of the ovarian hydrobursitis syndrome in dromedaries. Small sized ovarian hydrobursitis (⬍5 cm in diameter) could be medically treated.
Acknowledgments This study was supported by the Deanship for Scientific Research, Qassim University, Saudi Arabia.
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