Ovarian hydrobursitis in female camels (Camelus dromedaries): Biochemical, bacterial and protozoal evaluation

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Theriogenology 75 (2011) 734 –741 www.theriojournal.com

Ovarian hydrobursitis in female camels (Camelus dromedaries): Biochemical, bacterial and protozoal evaluation A. Alia,*, F. A. Al-Sobayila, M. Tharwata, K.M. Hassaneinb 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 24 July 2010; received in revised form 9 October 2010; accepted 10 October 2010

Abstract The aim of this study was to evaluate female camels affected with ovarian hydrobursitis (n ⫽ 31) for hematological and biochemical findings and for bacterial and protozoal infections. Blood samples were obtained and surgical ablation of the affected bursa was performed. Bursal fluid, follicular fluid, and serum were subjected to hormonal and biochemical analyses. Bursal fluids were cultured and colonies were identified using BioMérieux Vitek two compact system. Passive haemagglutination test was used for detection of Trypanosoma evansi. Indirect ELISA technique was carried out for detection of anti-Hydatid cysts anti-bodies. Neutrophilia was found in the affected animals (P ⫽ 0.01) with tendencies for monocytosis (P ⫽ 0.06) and eosinophelia (P ⫽ 0.05). Bursal fluid had a tendency for high estradiol-17␤ concentration compared to blood serum (P ⫽ 0.07). Progesterone and cholesterol concentrations were similar in bursal fluid, follicular fluid and serum. Total protein, phosphorus, and magnesium concentrations were greater (P ⬍ 0.05) in the bursal fluid than in serum. Oligella urethralis, Alloiococcus otitis, Granulicatella adicens, Escherichia coli, Sphingobacterium thalpophilum, Streptococcus sanguinis, Aeromonas salmonicida, Pseudomonas stutzeri, Staphylococcus warneri, Staphylococcus hominis, and Rhizobium radiobacter were isolated from 46.7% of bursal fluids. T. evansi was positive in 9.7% of cases. None were positive for hydatid cyst. Accordingly, we suggest that the ovarian hydrobursitis syndrome is initially an inflammatory process and the accumulated bursal fluid is partially originated from follicular fluid. © 2011 Elsevier Inc. All rights reserved. Keywords: Ovarian hydrobursitis; Camel; Blood analysis; Bacteria; Trypanosoma

1. Introduction In the female camels, the ovary is entirely enclosed within a fold of the mesosalpinx known as the ovarian bursa, the apex of this bursa forms a large circular orifice within which lies the fimbriae of the oviduct [1]. Ovarian hydrobursitis is a peculiar disease of the ovarian bursa, characterized by adhesion of the ovarian

* Corresponding author. Tel.: 0020882295574; fax: 0020882333938. E-mail address: [email protected] (A.M.H. Ali). 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.10.014

bursa, fluid accumulation, and encapsulation of the ovary [2,3]. Pathogenesis of the disease is still not clear and its association with other disease processes remains to be studied. A possible relationship between recurrent anovulatory hemorrhagic follicles and bursal adhesions has been suggested [3]. Echinoeoccus infestation has been identified in female camels affected with the ovarian hydrobursitis [4]; however, the role of this involvement remains unclear. Moreover, bacterial infection has been claimed as a primary cause of the disease [5].

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Additionally, presence of a genetic predisposition has been assumed for occurrence of the disease [3]. This syndrome is usually associated with long standing infertility, early embryonic death, and abortion [3]. The condition has been suspected on palpation, when difficulty has been encountered during examination and retraction of the uterus and ovaries [3,5]. Ultrasonography has been proved as a more reliable diagnostic technique and presented variable appearances of the ovarian-bursa depending on the size, ovarian activity and the nature of the fluid within the bursa [3]. Hormonal and biochemical evaluation of the bursal fluid and its comparison with blood and follicular fluid biochemistry may be useful in clarifying the origin of the bursal fluid. Moreover, bacterial and protozoal examination may be helpful in explaining the etiopathogenesis of the syndrome. The aim of the current study was, therefore, to examine female camels affected with ovarian hydrobursitis for bursal, follicular and blood constituents as well as for bacterial and protozoal infections. 2. Materials and methods 2.1. Animals and clinical examinations A total of 188 Arabian female camels (age from 5 to 14 years) were examined at the Veterinary Teaching Hospital of Qassim University, Saudi Arabia, during the breeding season (September 2009 to March 2010) for different etiologies of infertility. They were examined for general health condition (rectal temperature, heart and respiratory rates, ruminal movement, mucus membrane and lymph nodes) and for body condition score using a scale from 1 to 5 [6]. Gynecological examination was performed using the standard transrectal, vaginal, and ultrasonographic techniques (Aloka SSD-500, equipped with 5 or 7 MHz linear-array transducers, Aloka Co., Ltd., Tokyo, Japan). Of the examined animals, ovarian hydrobursitis was diagnosed in 31 of the 188 infertile cases (16.5%). The condition was suspected on trans-rectal palpation, when a difficulty was encountered during retraction of the uterus. A cord-like structure was palpated when passing the operator’s hand from the uterine horn to the corresponding ovary, this band extended forward and downward for a variable distance. At the end of the stretched band, the encapsulated bursa was mostly palpated as a round fluctuated sac with a difficulty in reaching or grasping the corresponding ovary. Ultrasonographically, the bursal fluid was mostly seen as a nonechogenic structure

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and the encapsulated ovary appeared as a floating echogenic mass within the bursal fluid. 2.2. Blood samples Two types of blood samples were drawn from the jugular vein of the 31 affected animals and from 10 normally cyclic non-affected animals (controls) at the time of clinical examination between 10 and 12 h AM, one on EDTA and the other in plane tubes for serum harvesting. Sampling of the controls was coincident with that of affected animals. 2.3. Hematological examinations The whole blood sample was used for estimation of hematocrite and total and differential white blood cell counts, red blood cell counts, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, and platelet count within 30 min of collection using the standard hematological techniques [7]. 2.4. Hormonal and biochemical analyses Serum was separated by centrifugation for 15 min at 1200⫻g and was immediately frozen at ⫺20 °C for future analyses. Surgical ablation of the affected bursa was performed in 15 affected cases, where bursal fluid was obtained [3]. When the encapsulated ovary was active (n ⫽ 8 cases), follicular fluids were collected from all follicles ⬎ 10 mm in diameters and pooled for each animal. Serum, bursal and follicular fluids were analyzed for concentrations of estradiol-17␤ and progesterone by ELISA (Bioteck ELX-800 reader, BioTek Insruments Inc., Germany) using commercial kits (Human Gesellschaft fur Biochemica und Diagnostica, Wiesbaden, Germany). ELISA technique is based on competitive interaction of progesterone/estradiol and the hormone-enzyme conjugate for a limited number of immobilized anti-progesterone/estradiol antibodies. Thus the amount of bound hormone-enzyme conjugate is inversely proportional to the concentration of hormones in the specimen. The coefficient of variance of intra- and interassay were 4.2 and 7.3% and 4.5 and 8.3%, and the sensitivity of the assay was 3 pgml⫺1 and 0.03 ng ml⫺1, for the estradiol-17␤ and progesterone, respectively. Moreover, bursal fluid, follicular fluid, and serum were photometrically analyzed for concentrations of sodium, potassium, chloride, calcium, phosphorus, magnesium, iron, total protein, albumin, cholesterol, triglyceride, blood urea nitrogen, creatinine, and aspartate aminotransferase using commercial kits provided by Human mbH.

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2.5. Bacteriological examination Bursal fluid specimens (n ⫽ 15) were aseptically collected from the affected bursa directly after surgical ablation using 20 mL sterile syringe. In the bacteriological lab, the collected specimens were cultured in two ways. Firstly, part of the specimens was streaked onto 10% sheep blood agar, Chocolate agar, MacConkey agar, mannitol salt agar and brain heart infusion agar (Medical Disposable Manufacturing Co., KSA, Riyadh) and incubated at 37 °C for 5 days. The cultures were checked daily for observation of bacterial growth. Secondly, 10 mL of the collected bursal fluid was cultured anaerobically onto BioMérieux BacT/ TALERT® 3D 60 system (Ref; 259793) up to 7 days before discarded as negative. The positive bottles were subcultured anaerobically on the cultured media mentioned above. The appeared colonies were identified by their colonial morphology, Gram’s stain and BioMérieux Vitek two compact system (Version 03.01 software) using BioMérieux Vitek two compact system identification cards. Relationship between physical characters of the obtained bursal fluid (amount, color, and consistency) and probability of bacterial isolation was investigated. Physical character of the bursal fluids were numerically scored (amount: ⬍ 1000 mL ⫽ 1, 1000 –3000 ⫽ 2, ⬎3000 mL ⫽ 3; color: transparent ⫽ 1, light colored ⫽ 2, deeply colored ⫽ 3; viscosity: watery ⫽ 1, viscous ⫽ 2, semisolid ⫽ 3) and correlated with the probability of bacterial isolation. 2.6. Serological test for the detection of Trypanosoma evansi Passive haemaggluination test was used to investigate the presence of Trypanosoma evansi antibodies in sera of affected female camels (n ⫽ 31) using technique described by Omar et al [8]. Briefly, serum samples were diluted in Tris buffer solution, pH 8.0. Each serum sample was tested using the microtitre technique. Twofold dilutions of sera from 1:8 to 1:14096 were made in V-bottomed microtitre olates (Greiner, Germany). After dilution the T. evansi /red blood suspension was added and inoculated at room temperature for 3hr. Sera from uninfected camel and an infected camel were used as negative and positive controls, respectively. Samples showing agglutination at 1:16 were considered positive. 2.7. Serological test for the detection of Hydatid cyst Indirect ELISA technique was used to investigate presence of anti-Hydatid cysts anti-bodies in sera of the affected female camels (n ⫽ 31) at a dilution of 1:40

[9]. Briefly, fertile Hydatid cysts fluid antigen (FHCFA) was prepared according to Ito et al [10]. Each plate was coated with the antigen at its optimal concentration (2 ␮g/ml coating buffer adjusted after checkerboard titration). Peroxidase-conjugated protein A (Sigma Aldrich Corp®, St. Louis, MO, USA) was used at 1:2000 dilution. Ortho-phenylenediamidine-OPD (Sigma Aldrich Corp®, St. Louis, MO, USA) was added at a concentration of 340 ug/ml substrate buffer. Absorbance was read at 492 nm using a Titerteck multiskan ELISA reader (Labsystem, ICN Biomedicals Multiskan Plus 311 BO, Ontario, Canada). Known positive and negative camel sera were used as control for the test. 2.8. Statistical analyses The variances were subjected to testing of their normal distribution using Kolmogorov-Smirnov test of the SPSS program, version 16, 2007 [11]. An analysis of variance (ANOVA) was used for comparison between treatment groups (serum vs bursal fluid vs follicular fluid) regarding the hormonal and biochemical concentrations. When a significant difference was recorded, the LSD was used to determine how the means differed. The ␹2-test was used to compare the same groups for hematocrit values. The t-test was used to compare sera of the affected and control animals for the hormonal and biochemical concentrations. Correlation coefficient was used to determine the relationship between physical characters of the bursal fluid and probability of bacterial isolation. Significant differences were set at P ⬍ 0.05 and the SPSS-program, version 16.0 [11] was used for the analyses. 3. Results 3.1. Clinical findings, breeding histories, and general health conditions Of the examined 188 infertile cases, endometritis, hydrobursitis, vaginal adhesion, ovarian inactivity, and ovarian cysts were diagnosed in frequencies of 118 (62.8%), 31 (16.5%), 22 (11.7%), 10 (5.3%), and 7 (3.7%), respectively. Cases with hydrobursitis (n ⫽ 31; 12 nullipara and 19 multipara) had body condition score ranged from 2 to 4. They had histories of reproductive disorders (refuse mating n ⫽ 5; repeat breeding with long heat interval n ⫽ 22, abortion n ⫽ 3, nymphomania n ⫽ 1) for a period ranging from 6 months to 4 years. At submission, none of these animals showed fever or reducing appetite. Heart and respiratory rates

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Table 1 Hematological findings in female camels affected with ovarian hydrobursitis (n ⫽ 31) compared to controls (n ⫽ 10). Blood parameter

Ovarian bursitis

Controls

P value

White blood cell (⫻109/L) Neutrophils (⫻109/L) Lymphocytes (⫻109/L) Monocytes (⫻109/L) Eosinophils (⫻109/L) Basophils (⫻109/L) Red blood cells (⫻1012/L) Hemoglobin (g/L) Hematocrit (%) Mean Corpuscular Volume (fl) Mean Corpuscular Hemoglobin (pg) Mean Corpuscular Hemoglobin Concentration (g/dl) Platelet count (⫻109/L)

16.5 ⫾ 4.5a 12.5 ⫾ 1.2a 3.5 ⫾ 1.0a 0.4 ⫾ 0.2a 0.4 ⫾ 0.1a 0.1 ⫾ 0.02a 13.3 ⫾ 3.71a 16.8 ⫾ 0.9a 33.4a 28.0 ⫾ 0.6a 13.0 ⫾ 0.5a 43.9 ⫾ 0.6a 481.5 ⫾ 465a

12.2 ⫾ 3.0a 7.9 ⫾ 1.8b 3.8 ⫾ 1.8b 0.2 ⫾ 0.1a 0.2 ⫾ 0.1a 0.1 ⫾ 0.03a 12.8 ⫾ 2.02a 16.3 ⫾ 0.5a 32.4a 27.5 ⫾ 1.0a 13.2 ⫾ 0.3a 49.6 ⫾ 4.0a 356.7 ⫾ 243a

0.2 0.01 0.5 0.06 0.05 0.7 0.9 0.4 0.3 0.2 0.5 0.09 0.3

Values in means ⫾ SEM. Values with different letters in the same row differ significantly.

as well as ruminal movements were within the normal values. Lymph nodes were not enlarged. 3.2. Hematological findings Complete blood count (CBC) in the female camels affected with ovarian hydrobursitis compared to controls is shown in Table 1. The mean white blood cell count was non-significantly greater in the affected group (P ⫽ 0.2). Moreover, there was a significant increase in the number of neutrophils in the affected group (P ⫽ 0.01) with tendencies for monocytosis (P ⫽ 0.06) and eosinophelia (P ⫽ 0.05). 3.3. Hormonal and biochemical findings Hormonal and biochemical constituents of the bursal fluid, follicular fluid, and serum of female camels affected with ovarian hydrobursitis are shown

in Table 2. Bursal fluid had high estradiol-17␤ concentration. Although, this concentration was significantly lower than that of the follicular fluid (P ⫽ 0.001), it is non-significantly greater than that of serum (P ⫽ 0.07). Progesterone and cholesterol concentrations did not differ among bursal fluid, follicular fluid, and serum. The total protein, phosphorus, and magnesium concentrations were greater in the bursal fluid than in serum. Though, these concentrations did not significantly differ between bursal and follicular fluids. The iron concentration was significantly greater in the follicular fluid than in bursal fluid or serum. The concentrations of the other tested parameters were similar in the bursal fluid, follicular fluid and serum. Hormonal and biochemical constituents of serum of the affected animals in comparison to controls are

Table 2 Hormonal and biochemical compositions of the bursal fluid (n ⫽ 15), follicular fluid (n ⫽ 8) and serum (n ⫽ 15) of female camels affected with ovarian hydrobursitis. Parameter

Bursal fluid

Follicular fluid

Serum

P value

Estradiol ⫺17 ␤ (pg ml⫺1) Progesterone (ng ml⫺1) Cholesterol (mg/dl) Total protein (g/dl) Albumin (g/dl) Calcium (mg/dl) Phosphorus (mg/dl) Magnesium (mg/dl) Iron (␮g/dl) Sodium (mmol/l) Postassium (mmol/l) Chloride (mmol/l)

442.5 ⫾ 200.0a 1.7 ⫾ 0.9a 96.2 ⫾ 12.0a 9.6 ⫾ 0.8a 4.3 ⫾ 0.4a 9.3 ⫾ 1.0a 8.8 ⫾ 2.0a 2.9 ⫾ 0.2a 323.1 ⫾ 85.0a 183.6 ⫾ 19.0a 4.2 ⫾ 0.5a 121.8 ⫾ 3.0a

1280.0 ⫾ 282.0b 4.3 ⫾ 1.0a 82.4 ⫾ 17.0a 8.3 ⫾ 1.0a 4.2 ⫾ 0.5a 9.6 ⫾ 1.0a 10.2 ⫾ 1.0a 2.6 ⫾ 0.2ab 675.0 ⫾ 112.0b 204.3 ⫾ 21.0a 5.5 ⫾ 0.5a 123.5 ⫾ 3.0a

117.0 ⫾ 100.0b 1.2 ⫾ 2.0a 81.2 ⫾ 6.0a 7.4 ⫾ 0.4b 4.2 ⫾ 0.2a 9.0 ⫾ 0.5a 6.30 ⫾ 0.6b 2.40 ⫾ 0.1b 194.3 ⫾ 43.0a 179.3 ⫾ 8.0a 4.1 ⫾ 0.2a 123.8 ⫾ 1.0a

0.001 0.1 0.6 0.04 0.9 0.9 0.02 0.01 0.001 0.5 0.06 0.7

Values in means ⫾SEM. Values with different letters in the same row differ significantly.

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Table 3 Hormonal and biochemical findings in serum of female camels affected with ovarian hydrobursittis (n ⫽ 31) compared to controls (n ⫽ 10). Parameter

Ovarian hydrobursitis

Control

P value

Estradiol ⫺17 ␤ (pg ml⫺1) Progesterone (ng ml⫺1) Total protein (g/dl) Albumin (g/dl) Urea (mg/dl) Cholesterol (mg/dl) Calcium (mg/dl) Phosphorus (mg/dl) Magnesium (mg/dl) Triglycerides (mg/dl) Creatinine (mg/dl) Aspartate aminotransferase (␮/l) Iron (␮g/dl) Sodium (mmol/l) Postassium (mmol/l) Chloride (mmol/l)

117.5 ⫾ 23.0a 2.5 ⫾ 0.5a 7.4 ⫾ 0.3a 4.2 ⫾ 0.2a 50.6 ⫾ 3.0a 81.2 ⫾ 6.0a 9.0 ⫾ 0.2a 6.3 ⫾ 0.3a 2.4 ⫾ 0.1a 42.7 ⫾ 2.0a 1.4 ⫾ 0.1a 4.1 ⫾ 1.0a 194.1 ⫾ 25.0a 179.0 ⫾ 0.6a 4.10 ⫾ 0.1a 123.8 ⫾ 1.0a

217.5 ⫾ 108.0a 3.8 ⫾ 0.7a 7.9 ⫾ 0.1a 4.4 ⫾ 0.1a 56.6 ⫾ 8.0a 81.3 ⫾ 4.0a 8.9 ⫾ 0.1a 6.9 ⫾ 0.8a 2.4 ⫾ 0.1a 41.5 ⫾ 3.0a 1.2 ⫾ 0.3.a 5.2 ⫾ 2.0a 185.7 ⫾ 32.0a 172.2 ⫾ 1.0a 3.8 ⫾ 0.1a 120.0 ⫾ 5.0a

0.2 0.08 0.5 0.3 0.9 0.4 0.2 0.6 0.5 0.4 0.6 0.8 0.5 0.3 0.1 0.4

Values in means ⫾ SEM. Values with different letters in the same row differ significantly.

shown in Table 3. There were no significant differences between groups for any of the included parameters. Different bacterial isolates (n ⫽ 11) were obtained from 7/15 (46.7%) of bursal fluids (Table 4). The others 8 cases showed no bacterial growth. Mixed (n ⫽ 4) and pure (n ⫽ 3) cultures were observed. There was no

relationship between physical characters of the bursal fluid and the probability of bacterial isolation. Out of 31 camels tested, three (9.7%) were found positive for T. evansi when screened with the passive haemagglutination test. One of these cases had bilateral ovarian hydrobursitis, the second showed complete ad-

Table 4 Bacterial isolates obtained from bursal fluids of female camels affected with ovarian hydrobursitis in relation to animal history and physical character of bursal fluid (n ⫽ 15). Animal

History

Parity

Age

1

Nulliparous

5y

2 3 4 5 6

Multiparous Multiparous Nulliparous Nulliparous Nulliparous

10 8 4 6 6

7

Multiparous

10 y

Repeat breeding

2y

8 9

Multiparous Multiparous

7y 12 y

Nympho-manic Abortion

2y 4y

10 11 12 13 14 15

Multiparous Multiparous Multiparous Nulliparous Nulliparous Nulliparous

8 8 8 5 7 5

Repeat breeding Repeat breeding Repeat breeding Repeat breeding Repeat breeding Abortion

3y 3y 3y 1y 2y 1y

y y y y y

y y y y y y

Complaint

Bursal fluid

No

Bacterial isolates

Duration

Amount

Color

Refuse mating

6 mo

800 mL

Transparent

Watery

Repeat Repeat Repeat Repeat Repeat

3y 2y 6 mo 1y 1y

Light brownish Black Deeply brownish Light brownish Brownish

Viscous Viscous Viscous Viscous Viscous

2000 mL

Chocolate

Viscous

1000 mL Cheesy content⫹scanty fluid 3400 mL 500 mL 2000 mL 100 mL 3000 mL 2500 mL

Straw like Brownish

Watery Semi-solids

Reddish Black Black Transparent Black Brownish

Watery Viscous Viscous Watery Viscous Viscous

breeding breeding breeding breeding breeding

3800 mL 4000 mL 1500 mL 2600 mL 120 mL ⫹ 150 mL (bilateral)

Viscosity Oligella urethralis, Alloiococcus otitis Granulicatella adicens Escherichia coli Negative Negative Sphingobacterium thalpophilum, Streptococcus sanguinis Aeromonas salmonicida, Pseudomonas stutzeri Negative Staphylococcus warneri, Staphylococcus hominis Negative Negative Negative Negative Rhizobium radiobacter Negative

Animals number 1 and 13; 8 and 9; 10, 11 and 12 were presented from a same herd. Animal number 2 and 4 showed adhesion between uterus and peritoneum. Animal number 9 had a very thick-walled bursa. Animal number 13 revealed purulent vaginal discharge.

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hesion of the vagina, and the third revealed a large hemorrhagic cyst on the encapsulated ovary. All three cases were multiparous. They showed lower mean body condition score, lower mean concentrations of iron and triglycerides, but greater concentrations of blood urea nitrogen and aspartate aminotransferase when compared with females had ovarian hydrobursitis but were negative for T. evansi (2.0.0 ⫾ 0.0 vs 3.3 ⫾ 0.4; 164.7 ⫾ 88.0 vs 197.8 ⫾ 27.0 ␮g/dl; 39.3 ⫾ 0.3 vs 43.1 ⫾ 1.9 mg/dl; 57.0 ⫾ 6.6 vs 49.8 ⫾ 2.8 mg/dl; 5.2 ⫾ 1.1 vs 3.3 ⫾ 0.2 ␮g/l, respectively). Due to small number of cases, these differences did not reach significant values. None of female camels tested were positive for Hydatid cysts when screened by indirect ELISA technique. 4. Discussion This is the first report regarding the hormonal and biochemical constituents of the bursal content of female camels affected with ovarian hydrobursitis. Hormonal and biochemical evaluation of the bursal fluid and its comparison with blood and follicular fluid biochemistry was done in order to investigate the origin of the bursal fluid and help clarify the pathogenesis of the disease. The high concentration of estradiol-17␤ suggested that the bursal content is partially originated from follicular fluid. Supporting this opinion, concentrations of certain biochemical parameters—like phosphorus and magnesium—were similar in the bursal and follicular fluids. Additionally, progesterone and cholesterol concentrations were similar in the bursal fluid, follicular fluid and serum. Accumulation of fluids due to rupture of the ovarian follicles is highly probable in female camels [3]. Anatomically, the mesosalpinx, mesovarium and proper ligament of the ovary are joined together that makes the ovary of camels completely hidden within the ovarian bursa [12,13]. Moreover, it has been suggested that encapsulation of the ovary and collection of fluid from successive rupture of hemorrhagic follicles may result in formation of ovario-bursal adhesion [3]. The values obtained from the camel blood chemistry were comparable with those values reported in the previous studies [14,15]. The biological variations due to animal age and parity did not influence the results. High total protein in the body fluids can be a warning sign of chronic inflammation or infection. An increase in serum total protein, largely attributable to increased ␥-globulin, has been noted in positively reacting cattle

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sera to Brucella abortus [16]. Moreover, increase in serum total protein has been also found in female camels affected with vaginal adhesions [17]. The high concentration of total protein in the bursal fluid observed in this study may be, therefore, an indicator for occurrence of inflammatory process in the bursal content and/or bursal wall. Some of the obtained follicular fluids were hemorrhagic; hence the iron concentration was greater in the follicular fluid than in serum or bursal fluid. Neutrophils are typically considered the first line of cellular defense against pathogens [7]. Thus, neutrophelia observed in this study additionally suggests that this syndrome may be originally an inflammatory process. Tendencies for monocytosis and eosinophelia may be attributed to the chronocity of the disease and for the parasitic infection. Microbiological examinations of the bursal content have been found negative in the study of Tibary and Anouassi [3], and positive in 70% of the cases included in the study of Al-Eknah and Ali [5]. In the current study, bacterial growth was observed in about half of the affected cases, where 11 different isolates were obtained. Some of the obtained bacteria have been previously isolated from aborted fetuses or infected reproductive tracts. Streptococcus sanguinis type 1 has been isolated in pure culture from lungs, spleen, liver, and stomach and in mixed culture with E. Coli from placental tissues of an aborted llama fetus [18]. Staphylococcus warneri has been reported as a possible cause of bovine abortion [19]. It has been isolated in pure culture from the lung, liver, and stomach contents of a bovine fetus at 5 months of gestation. Escherichia coli has been observed in cases with purulent vaginal discharges in female camels [20]. Aeromonas hydrophilla has been obtained from cases with ovarian hydrobursitis in female camels [5]. Oligella urethralis (formely Moraxella urethralis) has been also isolated from dairy cattle herd with cystitis, urethritis and pyelonephritis [21]. Staphylococcus hominis has been sampled from haircoat, nares, vagina, teat skin, and streak canal of heifers [22]. The other obtained bacteria of this study have not been previously isolated from the reproductive tract either in human or animals, thus we could not estimate its role in the current syndrome. Granulicatella adiacens has been associated with haematological malignancies, neutropenic fever, valvular problems, infective endocarditis, and ischemic heart disease in humans [23]. Pseudomonas stutzeri is widely distributed in the environment, and it has also been isolated as an oppor-

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tunistic pathogen from humans. Alloiococcus otitis is quite commonly isolated from middle ear infections (otitis media), both in humans and animals [24]. Sphingobacterium thalpophilum is a bacteria that belong to the genus Sphingobacterium and affects the respiratory tract of human [25]. Rhizobium radiobacter is uncommon opportunistic pathogen present in soil. It has been particularly associated with indwelling intravascular devices in immunocompromised patients [26]. Results showed that the probability of bacterial isolation could not be predicated from physical characters of the obtained bursal fluid. Activity of the encapsulated ovary might be the determinable factor for the amount, color and viscosity of the accumulated bursal fluid. Low fertility and abortion have been generally attributed to trypanosoma infection in camels [27]. Moreover, emaciation, anemia and disturbances of blood parameters have been commonly observed in affected camels [28,29]. In this study, three cases were positive for T. evansi that also showed low body condition score and disturbance in biochemical parameters. Hydatids, caused by the tapeworm belonging to the genous Echinococcus have been found in numerous mammalian species including humans. Hydatid cysts are frequently observed in the lung and liver [30]. In the study by Shalash and Nawito [4], 40% of the hydrobursitis cases were associated with Echinoeoccus infestation. In contrast, none of the included cases were serologically positive for Hydatid cyst. According to the obtain results (neutrophelia, tendencies for monocytosis and eosinophilia, high total protein, bacterial and protozoal infections), we suggest that the ovarian hydrobursitis syndrome is initially an inflammatory process. Due to many similarities between bursal and follicular fluids, we assume that the accumulated bursal fluid is partially follicular in origin.

Acknowledgments The authors wish to thank Dr M.M. El-Bahy, Prof of Parasitology and Dr O.H. Omar, Associate Professor of Laboratory Diagnosis, College of Agriculture and Veterinary Medicine, Qassim University, King Saudi Arabia, for their kind laboratory and scientific supports.

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