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Isolation of E. coli from foam and effects of fluoroquinolones on E. coli and foam production in male Japanese quail
Theriogenology 62 (2004) 1383–1390 www.elsevier.com/locate/theriogenology
Isolation of E. coli from foam and effects of fluoroquinolones on E. coli and foam production in male Japanese quail Jag Mohana,*, Kochiganti Venkata Hanumat Sastrya, Jagbir Singh Tyagia, Dhirendra Kumar Singhb a
Division of Physiology and Reproduction, Central Avian Research Institute, Izatnagar (U.P.) 243122, India Division of Veterinary Public Health, Indian Veterinary Research Institute Izatnagar (U.P.) 243122, India
b
Received 7 October 2003; accepted 15 December 2003
Abstract Sexually active male Japanese quail (Coturnix coutrnix Japonica) produce a foamy substance from their cloacal gland. It was postulated that bacteria played an important role in production of foam. The primary objective of this study was to isolate and identify bacteria present in the cloacal foam. The secondary objective was to evaluate the effect of fluoroquinolone treatment on bacterial counts and foam production. Healthy adult Japanese quail were maintained in individual cages under uniform husbandry conditions and allocated arbitrarily into three groups (each group consisted of 12 male and 12 female birds). Foam was collected from the cloacal gland of male birds of each group separately into sterile petri dishes and was cultured to isolate and identify bacteria and to determine their sensitivity to various antibiotics. Escherichia coli bacteria, sensitive to various antibacterials (including the fluoroquinolones ciprofloxacin and pefloxacin), were isolated. In the second part of the study, male quails of Group I (control) received I mL vehicle (normal saline 0.9% (w/v) NaCl) daily (via the intraperitoneal route) for 12 days. Male birds from groups II and III were treated intraperitoneally with ciprofloxacin or pefloxacin at the rate of 10 mg and 12 mg per/kg body weight respectively, for 12 days. In antibiotic-treated birds, there was a gradual reduction in foam production during treatment. At the end of treatment, the cloacal gland area was smaller (P < 0.05) in pefloxacin-treated birds compared to the other groups. Furthermore, a trend towards decreasing body weight and fertilizing ability was noted in the same group. A drastic reduction in bacterial counts of foam was recorded only in fluoroquinolone-treated groups during treatment period. After cessation of * Corresponding author. Tel.: +91 581 230 0204; fax: +91 581 230 1321. E-mail address: [email protected] (J. Mohan). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.12.033
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treatment, all end points were increasing back to pre-treatment levels. In conclusion, E. coli were present in the foam of the cloacal gland of Japanese quail and may have a role in foam production. # 2004 Elsevier Inc. All rights reserved. Keywords: Japanese quail; Fluoroquinolones; Foam; Cloacal gland; Fertility
1. Introduction Sexually active male Japanese quail (Coturnix coturnix Japonica) has a well-developed cloacal gland that is unique among birds. This gland produces a meringue-like foam that is released during mating and defecation [1]. During mating, the male Japanese quail empties the foamy substance into the vent of the female. Bigger glands were associated with larger testes, increased sexual activity and higher fertility in Japanese quail [2–5]. Mechanisms controlling foam synthesis by the cloacal gland have not been characterized. It was hypothesized that bacteria play an important role in the conversion of mucoid secretion of the cloacal gland into the meringue-like foam [6]. The bacteria metabolize glucose (and other sugars present in the secretion), resulting in the formation of carbon dioxide and hydrogen gas. These gases get impregnated into the mucoid secretion of the gland, giving the foamy consistency [6]. However, the type of bacteria involved in the foam synthesis is not known. If bacteria are involved in foam production, it is expected that an appropriate antimicrobial agent could eliminate them, thereby suppressing foam production. Fluoroquinolones are synthetic antibiotics used in preventive and therapeutic treatment against a variety of bacterial infections in farm animals and humans. One of the most commonly used fluoroquinolones is ciprofloxacin {1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1piperazinyl)-3-quinoline carboxylic acid}. This drug was primarily used to treat humans because of its spectrum and effectiveness against a wide range of bacteria, including gram negative bacteria, i.e. various species of Enterobacteriaceae and Pseudomonadaseae, some gram-positive bacteria, many aerobic and facultative anaerobic bacteria, as well as Mycoplasma and Rickettsia [7–11]. Another fluoroquinolone, pefloxacin {1-ethyl-6fluoro-1,4-dihydro-4-oxo-7-(4-methyl-1-piperazinyl)–quinoline-3-carboxylic acid (1589 RB)} is successfully employed in the treatment of infections caused by many gram positive and gram-negative bacteria [12–14]. Pefloxacin is also used in the treatment of serious infections caused by intracellular bacteria [15]. The bactericidal mechanism ciprofloxacin and pefloxacin is interference in bacterial DNA gyrase activity and DNA replication, similar to other quinolones [16,17]. The primary objective of this study was to isolate and identify bacteria present in the cloacal foam of Japanese quail. The secondary objective was to evaluate the effect of fluoroquinolone treatment on bacterial counts and foam production. 2. Materials and methods 2.1. Birds and experimental plan Mature male and female (36 of each gender) Japanese quails (heavy-body strain) were randomly selected at 12 week of age. The birds were reared and maintained under
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uniform husbandry conditions with an ambient temperature of 18 to 23 8C. All birds were allowed access to a standard breeder ration and water ad libitum with a constant light 14 h/day during the experimental period. At 18 week of age, male birds were randomly divided into three groups of 12 birds each and were placed in individual cages. Group I served as control; they were given normal saline {0.9% (w/v) NaCl} via the intraperitoneal route. The males of groups II and III received ciprofloxacin and pefloxacin at the rate of 10 mg/kg body weight and 12 mg/kg body weight respectively by the same route for the similar period. All treatments were done daily at 10:00 h until the foam production in antibiotic-treated birds appeared to stabilize (12 days). 2.2. Measurement of the area of the cloacal gland The area of cloacal gland of sexually active mature adult birds was measured with Vernier calipers. The product of the height (dorsoventral) and width (lateral) of the cloaca was used to calculate the area of the cloacal gland [18]. 2.3. Determination of foam production and body weight Foam was collected in sterile petri dishes by gently squeezing both sides of the cloacal gland of male Japanese quail at 12:00 h. Precautions were taken to avoid fecal contamination. Foam collection was done daily on Days 1–12, and then at 6-day intervals from Days 18–42. The foam was immediately weighed with an electronic balance [5]. Body weight of the birds was also determined with the electronic balance. 2.4. Sperm fertilizing ability To examine the fertilizing ability of all male birds, they were paired in suitable cages with an equal number of healthy females of the same hatch, so that each adult male was allowed to mate with one female [1:1]. After 2 week of pairing (just before treatment) eggs were collected from all females. Similarly, eggs were collected from female of each group during the middle of treatment (Day 6), end of treatment (Day 12) and 1 week after treatment (Day 19). Experimental eggs were stored, and the fertilizing ability of all males was subsequently evaluated [5]. 2.5. Total bacterial counts Bacterial counts in foam before, during and after withdrawal of treatment at different time interval was made by surface counting of viable bacteria [19]. Briefly, a ten-fold serial dilution of the foam in sterile peptone water was prepared and 10 mL from each dilution was inoculated on a sheep blood agar plate in triplicate. The plate was incubated overnight at 37 8C. At the end of incubation, the plates were taken out, colonies counted and results were presented as average colony forming units (cfu).
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Table 1 Antibiogram of isolates of foam produced from the cloacal gland of Japanese quail Antibiotics
Sensitivity pattern
Ciprofloxacin Pefloxacin Enrofloxacin Norfloxacin Gentamycin Oxytetracycline Chlorotetracycline Ampicillin Amikacin Novobiocin Lincomycin Erythromycin
+++ +++ +++ +++ +++ +++ +++ +++ +++ + R R
+++: Sensitive; +: Intermediate; R: Resistant.
2.6. Antibiogram The bacterial isolates from the foam produced by cloacal gland of Japanese quail were tested for their sensitivity to various antibiotics (Table 1) using the disc diffusion method on agar plates [20]. A nutrient agar plate was swabbed with broth culture of the foam isolate and the antibiotic discs were placed on it, 2 cm apart. The plate was incubated overnight at 37 8C. The next day, the zone of inhibition was measured and deemed sensitive, intermediate and resistant, according to the interpretation chart of the supplier (Hi-Media, Mumbai, India). 2.7. Isolation and Identification of bacteria Isolation and identification of bacteria from the foam was done according to the procedure of Edwards and Ewing [21]. Foam was inoculated on sheep blood agar and MacConkey agar plates, which were incubated at 37 8C. The next day, colonies with identical morphology on both the plates were detected (indicating a pure culture). Gram’s staining revealed gram’s negative small rods. Four colonies from each plate were then picked up and streaked on to an EMB agar plate, which produced a typical metallic sheen. Further, identification of the isolate was made on the basis of biochemical tests (i.e. oxidase, catalese, indole, methyl red, voges-proskauer, and citrate utilization [21]). The identification was repeated three times. 2.8. Statistical analysis Foam production, bacterial counts, and fertilizing ability were analysed with ANOVA for repeated measures. Body weight and the area of the cloacal gland were analysed with a paired t-test [22].
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3. Results 3.1. Antibiogram The sensitivity of the isolates to various antimicrobials is shown in Table 1. 3.2. Isolation and identification of bacteria The isolates obtained from the foam were consistently identified as Escherichia coli. 3.3. Bacterial counts Both antibiotics dramatically decreased bacterial counts, but these counts recovered after cessation of treatment (Fig. 1). 3.4. Foam production Both antibiotics caused a substantial decrease in foam production, with a gradual recovery towards pre-treatment amounts after cessation of treatment (Fig. 2). 3.5. Body weight and area of the cloacal gland Although there were no significant differences in body weight, the reduction in the pefloxacin-treated birds (9.9%) seemed larger than in the other two groups (Table 2). Furthermore, cloacal gland area was decreased (P < 0.05) in pefloxacin-treated birds.
Fig. 1. Effect of ciprofloxacin and pefloxacin on bacterial counts (mean S.E.M.) in foam synthesized by the cloacal gland of Japanese quail.
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Fig. 2. Effect of ciprofloxacin and pefloxacin on foam production (mean S.E.M.) in male Japanese quail. The arrow indicates the withdrawal of treatment, followed by a 6-day resting period during which no foam production was recorded. Subsequently, foam was collected at 6-day intervals.
Table 2 Effects of ciprofloxacin and pefloxacin on different variables of Japanese quail at start and end of experiment (Days 1 and 12, respectively; mean S.E.M., n = 12) Area of cloacal gland (mm2)
Body weight (g) Day
Control
Ciprofloxacin
Pefloxacin
Control
Ciprofloxaxin
Pefloxacin
1 12
185 7.4 175 8.6
193 8.6 178 7.2
181 6.2 163 8.1
338 10.8 304 13.5
374 9.4 329 17.0
340 6.1 284* 15.0
*
Difference (P < 0.05) between Days 1 and 12.
Table 3 Effects of ciprofloxacin and pefloxacin treatment on the fertilizing ability (%) of quail spermatozoa Day
Control
Ciprofloxacin
Pefloxacin
0 6 12 19
100 100 91 90
100 94 87 89
100 95 81 84
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3.6. Fertilizing ability At the end of treatment period, fertilizing ability was lowest in pefloxacin-treated males, but the difference was not significant (Table 3). After cessation of treatment, fertilizing ability appeared to be recovering.
4. Discussion We consistently isolated E. coli from the foam. A reduction in E. coli counts was recorded in the foam even 2 h after the first treatment (data not shown). It was reported that fluoroquinolones penetrate very fast into the bacteria [23], which may be responsible for inhibition of DNA gyrase activity, an essential enzyme involved in DNA replication of bacteria [16,17]. Fluoroquinolones, therefore, disrupt DNA function, leading to death of the bacteria, consistent with the decreased number of bacterial counts in birds that received antibiotic treatment. That the decrease in E. coli counts occurred at the same time as reduced foam production in antibiotic-treated birds suggested that the bacteria have a role in foam formation. Although this is the first report on the effect of fluoroquinolones on foam production, McFarland et al. [6] also hypothesized the presence of bacteria in cloacal gland of Japanese quail and postulated their role in foam synthesis. More studies are needed to clearly elucidate the role of bacteria in foam production. The decline in fertilizing ability, body weight and area of cloacal gland was more prominent in the pefloxacin-treated birds compared to the other groups. Recently, we reported that the area of cloacal gland was positively correlated with plasma testosterone concentration, body weight and fertilizing ability of male Japanese quail [5]. Further, regression of cloacal gland in male Japanese quail by cyproterone acetate administration suggested that the cloacal gland is androgen-dependent and involved in maintenance of fertility [24]. Therefore, a reduction in cloacal gland area in the pefloxacin group may be partly responsible for decline in fertility and body weight in this group. Alternatively, the drop in fertility in the present study might be due to a toxic effect of the antibiotics on spermatozoa as well as spermatogonia and a secondary effect on steroid hormone negative feedback via the testes on the hypothalamus. That pefloxacin caused greater suppression of cloacal gland area and a trend towards greater suppression of body weight and fertilizing ability may be due to the shorter half-life of ciprofloxacin compared to other fluoroquinolones [25]. In that regard, pefloxacin concentrations in the cloacal gland and other tissues (breast muscle, kidney) at different time intervals after treatment (1, 5 and 10 days were several-fold higher than ciprofloxacin concentrations (unpublished observations). In conclusion, E. coli were present in the foam of the cloacal gland of Japanese quail and may have a role in foam production.
References [1] Sachs BD. Photoperiodic control of the cloacal gland of the Japanese quail. Science 1967;157:201–3. [2] Coil WH, Wetherbee DK. Observations on the cloacal gland of Eurasian quail. Chil J Sci 1959;59:268–70.
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[3] Sachs BD. Photoperiodic control of reproductive behavior and physiology of the Japanese quail. Horm Behav 1969;1:7–24. [4] Mohan J, Moudgal RP, Narayan R, Singh J, Singh RV. Prediction of the fertility potential of male Japanese quail. In: Proceedings of the XXI World’s Poultry Congress. Montreal; 2000.22.06. [5] Mohan J, Moudgal RP, Sastry KVH. Singh J, Singh RV. Effects of hemicastration and castration on foam production and its relationship with fertility in male Japanese quail. Theriogenology 2002;58:29–39. [6] Mc Farland LZ, Warner RL, Wilson WO, Mather FB. Cloacal gland complex of the Japanese quail. Experentia 1968;24:941–3. [7] Bauernfeind A, Petermuller C. In vitro activity of ciprofloxacin, norfloxacin and nalidixic acid. Eur J Clin Microbiol 1983;2:111–5. [8] Fass RJ. In vitro activity of ciprofloxacin (Bay 09867). Antimicrob Agents Chemother 1983;24:568–74. [9] Wise R, Andrews JM, Edwards lJ.. In vitro activity of Bay 09867, a new quinolone derivative, compared with those of other antimicrobial agents. Antimicrob Agents Chemother 1983;23:559–64. [10] Chin N, Neu HC. Ciprofloxacin a quinoline carboxylic acid compound active against aerobic and anaerobic bacteria. Antimicrob Agents Chemother 1984;25:319–26. [11] Nouws JFM. Mevius DJ, Vree TB, Baars AM, Laurensen J. Pharmacokinetics, renal clearance and metabolism of ciprofloxacin following intravenous and oral administration to calves and pigs. Vet Q 1988;10:156–63. [12] Goueffon YG, Montay F, Roquet M, Pesson. A new synthetic antimicrobial agent: 4-dihydro-1-ethyl-6fluoro-7-(4methyl-1-piperazinyl)-4-oxoquinoline-3-carboxylic acid (1589 RB). Acad Sci 1981;292:30–7. [13] Thabaut A, Durosoir JL. In vitro antibacterial activity of pefloxacin, nalidixic acid, pipemidic acids and flumequin. Pathol Biol 1982;30:394–7. [14] Thibault M, Koumare B, Soussy CJ, Duval J. Structure-activity relationships in quinolone group: antibacterial activity study of two new compounds. Ann Microbiol Paris 1981;132A:267–81. [15] Leysen DC, Haemers A, Pattyn SR. Mycobacteria and the new quinolones. Antimicrob Agents Chemother 1989;33:1–5. [16] Cozarelli NR. DNA gyrase and the supercoiling of DNA. Science 1980;207:953–60. [17] Gellert M. DNA Topoisomerases. Annu Rev Biochem 1981;50:79–910. [18] Siopes TD, Wilson WO. The cloacal gland an external indicator of testicular development in coturnix. Poult Sci 1975;54:1225–9. [19] Miles AA, Misra SS. Method of counting bacteria by surface viable count. J Hyg London 1938;38:372. [20] Baur MR, Kingby WMM. Sherris JC, Truck M. Antibiotics susceptibility testing by a standard disk method. Am J Clin Pathol 1966;45:493–6. [21] Edwards PR, Ewing WH. Identification of Enterobacteriacae. 3rd ed. Minnesota, USA: Burgess Publishing Co. Minneapolis; 1972. [22] Snedecor GW, Cochran WG. Statistical Methods. USA: The Iowa State University Press Ames IA; 1967. [23] Piddock LJ. New quinolones and gram-positive bacteria. Antimicrob Agents Chemother 1994;38:163–9. [24] Mohan J, Sastry KVH. Tyagi JS. Effects of cyproterone acetate on cloacal gland and testicular functions in Japanese quail. Ind J Poult Sci 2002;37:247–51. [25] Garcia Ovando H, Gorla N, Luders C, Poloni G, Errecalde C, Prieto G, Puelles I. Comparative pharmacokinetics of enrofloxacin and ciprofloxacin in chickens. J Vet Pharmacol Therap 1999;22:209–12.
Isolation of E. coli from foam and effects of fluoroquinolones on E. coli and foam production in male Japanese quail Jag Mohana,*, Kochiganti Venkata Hanumat Sastrya, Jagbir Singh Tyagia, Dhirendra Kumar Singhb a
Division of Physiology and Reproduction, Central Avian Research Institute, Izatnagar (U.P.) 243122, India Division of Veterinary Public Health, Indian Veterinary Research Institute Izatnagar (U.P.) 243122, India
b
Received 7 October 2003; accepted 15 December 2003
Abstract Sexually active male Japanese quail (Coturnix coutrnix Japonica) produce a foamy substance from their cloacal gland. It was postulated that bacteria played an important role in production of foam. The primary objective of this study was to isolate and identify bacteria present in the cloacal foam. The secondary objective was to evaluate the effect of fluoroquinolone treatment on bacterial counts and foam production. Healthy adult Japanese quail were maintained in individual cages under uniform husbandry conditions and allocated arbitrarily into three groups (each group consisted of 12 male and 12 female birds). Foam was collected from the cloacal gland of male birds of each group separately into sterile petri dishes and was cultured to isolate and identify bacteria and to determine their sensitivity to various antibiotics. Escherichia coli bacteria, sensitive to various antibacterials (including the fluoroquinolones ciprofloxacin and pefloxacin), were isolated. In the second part of the study, male quails of Group I (control) received I mL vehicle (normal saline 0.9% (w/v) NaCl) daily (via the intraperitoneal route) for 12 days. Male birds from groups II and III were treated intraperitoneally with ciprofloxacin or pefloxacin at the rate of 10 mg and 12 mg per/kg body weight respectively, for 12 days. In antibiotic-treated birds, there was a gradual reduction in foam production during treatment. At the end of treatment, the cloacal gland area was smaller (P < 0.05) in pefloxacin-treated birds compared to the other groups. Furthermore, a trend towards decreasing body weight and fertilizing ability was noted in the same group. A drastic reduction in bacterial counts of foam was recorded only in fluoroquinolone-treated groups during treatment period. After cessation of * Corresponding author. Tel.: +91 581 230 0204; fax: +91 581 230 1321. E-mail address: [email protected] (J. Mohan). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.12.033
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treatment, all end points were increasing back to pre-treatment levels. In conclusion, E. coli were present in the foam of the cloacal gland of Japanese quail and may have a role in foam production. # 2004 Elsevier Inc. All rights reserved. Keywords: Japanese quail; Fluoroquinolones; Foam; Cloacal gland; Fertility
1. Introduction Sexually active male Japanese quail (Coturnix coturnix Japonica) has a well-developed cloacal gland that is unique among birds. This gland produces a meringue-like foam that is released during mating and defecation [1]. During mating, the male Japanese quail empties the foamy substance into the vent of the female. Bigger glands were associated with larger testes, increased sexual activity and higher fertility in Japanese quail [2–5]. Mechanisms controlling foam synthesis by the cloacal gland have not been characterized. It was hypothesized that bacteria play an important role in the conversion of mucoid secretion of the cloacal gland into the meringue-like foam [6]. The bacteria metabolize glucose (and other sugars present in the secretion), resulting in the formation of carbon dioxide and hydrogen gas. These gases get impregnated into the mucoid secretion of the gland, giving the foamy consistency [6]. However, the type of bacteria involved in the foam synthesis is not known. If bacteria are involved in foam production, it is expected that an appropriate antimicrobial agent could eliminate them, thereby suppressing foam production. Fluoroquinolones are synthetic antibiotics used in preventive and therapeutic treatment against a variety of bacterial infections in farm animals and humans. One of the most commonly used fluoroquinolones is ciprofloxacin {1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1piperazinyl)-3-quinoline carboxylic acid}. This drug was primarily used to treat humans because of its spectrum and effectiveness against a wide range of bacteria, including gram negative bacteria, i.e. various species of Enterobacteriaceae and Pseudomonadaseae, some gram-positive bacteria, many aerobic and facultative anaerobic bacteria, as well as Mycoplasma and Rickettsia [7–11]. Another fluoroquinolone, pefloxacin {1-ethyl-6fluoro-1,4-dihydro-4-oxo-7-(4-methyl-1-piperazinyl)–quinoline-3-carboxylic acid (1589 RB)} is successfully employed in the treatment of infections caused by many gram positive and gram-negative bacteria [12–14]. Pefloxacin is also used in the treatment of serious infections caused by intracellular bacteria [15]. The bactericidal mechanism ciprofloxacin and pefloxacin is interference in bacterial DNA gyrase activity and DNA replication, similar to other quinolones [16,17]. The primary objective of this study was to isolate and identify bacteria present in the cloacal foam of Japanese quail. The secondary objective was to evaluate the effect of fluoroquinolone treatment on bacterial counts and foam production. 2. Materials and methods 2.1. Birds and experimental plan Mature male and female (36 of each gender) Japanese quails (heavy-body strain) were randomly selected at 12 week of age. The birds were reared and maintained under
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uniform husbandry conditions with an ambient temperature of 18 to 23 8C. All birds were allowed access to a standard breeder ration and water ad libitum with a constant light 14 h/day during the experimental period. At 18 week of age, male birds were randomly divided into three groups of 12 birds each and were placed in individual cages. Group I served as control; they were given normal saline {0.9% (w/v) NaCl} via the intraperitoneal route. The males of groups II and III received ciprofloxacin and pefloxacin at the rate of 10 mg/kg body weight and 12 mg/kg body weight respectively by the same route for the similar period. All treatments were done daily at 10:00 h until the foam production in antibiotic-treated birds appeared to stabilize (12 days). 2.2. Measurement of the area of the cloacal gland The area of cloacal gland of sexually active mature adult birds was measured with Vernier calipers. The product of the height (dorsoventral) and width (lateral) of the cloaca was used to calculate the area of the cloacal gland [18]. 2.3. Determination of foam production and body weight Foam was collected in sterile petri dishes by gently squeezing both sides of the cloacal gland of male Japanese quail at 12:00 h. Precautions were taken to avoid fecal contamination. Foam collection was done daily on Days 1–12, and then at 6-day intervals from Days 18–42. The foam was immediately weighed with an electronic balance [5]. Body weight of the birds was also determined with the electronic balance. 2.4. Sperm fertilizing ability To examine the fertilizing ability of all male birds, they were paired in suitable cages with an equal number of healthy females of the same hatch, so that each adult male was allowed to mate with one female [1:1]. After 2 week of pairing (just before treatment) eggs were collected from all females. Similarly, eggs were collected from female of each group during the middle of treatment (Day 6), end of treatment (Day 12) and 1 week after treatment (Day 19). Experimental eggs were stored, and the fertilizing ability of all males was subsequently evaluated [5]. 2.5. Total bacterial counts Bacterial counts in foam before, during and after withdrawal of treatment at different time interval was made by surface counting of viable bacteria [19]. Briefly, a ten-fold serial dilution of the foam in sterile peptone water was prepared and 10 mL from each dilution was inoculated on a sheep blood agar plate in triplicate. The plate was incubated overnight at 37 8C. At the end of incubation, the plates were taken out, colonies counted and results were presented as average colony forming units (cfu).
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Table 1 Antibiogram of isolates of foam produced from the cloacal gland of Japanese quail Antibiotics
Sensitivity pattern
Ciprofloxacin Pefloxacin Enrofloxacin Norfloxacin Gentamycin Oxytetracycline Chlorotetracycline Ampicillin Amikacin Novobiocin Lincomycin Erythromycin
+++ +++ +++ +++ +++ +++ +++ +++ +++ + R R
+++: Sensitive; +: Intermediate; R: Resistant.
2.6. Antibiogram The bacterial isolates from the foam produced by cloacal gland of Japanese quail were tested for their sensitivity to various antibiotics (Table 1) using the disc diffusion method on agar plates [20]. A nutrient agar plate was swabbed with broth culture of the foam isolate and the antibiotic discs were placed on it, 2 cm apart. The plate was incubated overnight at 37 8C. The next day, the zone of inhibition was measured and deemed sensitive, intermediate and resistant, according to the interpretation chart of the supplier (Hi-Media, Mumbai, India). 2.7. Isolation and Identification of bacteria Isolation and identification of bacteria from the foam was done according to the procedure of Edwards and Ewing [21]. Foam was inoculated on sheep blood agar and MacConkey agar plates, which were incubated at 37 8C. The next day, colonies with identical morphology on both the plates were detected (indicating a pure culture). Gram’s staining revealed gram’s negative small rods. Four colonies from each plate were then picked up and streaked on to an EMB agar plate, which produced a typical metallic sheen. Further, identification of the isolate was made on the basis of biochemical tests (i.e. oxidase, catalese, indole, methyl red, voges-proskauer, and citrate utilization [21]). The identification was repeated three times. 2.8. Statistical analysis Foam production, bacterial counts, and fertilizing ability were analysed with ANOVA for repeated measures. Body weight and the area of the cloacal gland were analysed with a paired t-test [22].
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3. Results 3.1. Antibiogram The sensitivity of the isolates to various antimicrobials is shown in Table 1. 3.2. Isolation and identification of bacteria The isolates obtained from the foam were consistently identified as Escherichia coli. 3.3. Bacterial counts Both antibiotics dramatically decreased bacterial counts, but these counts recovered after cessation of treatment (Fig. 1). 3.4. Foam production Both antibiotics caused a substantial decrease in foam production, with a gradual recovery towards pre-treatment amounts after cessation of treatment (Fig. 2). 3.5. Body weight and area of the cloacal gland Although there were no significant differences in body weight, the reduction in the pefloxacin-treated birds (9.9%) seemed larger than in the other two groups (Table 2). Furthermore, cloacal gland area was decreased (P < 0.05) in pefloxacin-treated birds.
Fig. 1. Effect of ciprofloxacin and pefloxacin on bacterial counts (mean S.E.M.) in foam synthesized by the cloacal gland of Japanese quail.
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Fig. 2. Effect of ciprofloxacin and pefloxacin on foam production (mean S.E.M.) in male Japanese quail. The arrow indicates the withdrawal of treatment, followed by a 6-day resting period during which no foam production was recorded. Subsequently, foam was collected at 6-day intervals.
Table 2 Effects of ciprofloxacin and pefloxacin on different variables of Japanese quail at start and end of experiment (Days 1 and 12, respectively; mean S.E.M., n = 12) Area of cloacal gland (mm2)
Body weight (g) Day
Control
Ciprofloxacin
Pefloxacin
Control
Ciprofloxaxin
Pefloxacin
1 12
185 7.4 175 8.6
193 8.6 178 7.2
181 6.2 163 8.1
338 10.8 304 13.5
374 9.4 329 17.0
340 6.1 284* 15.0
*
Difference (P < 0.05) between Days 1 and 12.
Table 3 Effects of ciprofloxacin and pefloxacin treatment on the fertilizing ability (%) of quail spermatozoa Day
Control
Ciprofloxacin
Pefloxacin
0 6 12 19
100 100 91 90
100 94 87 89
100 95 81 84
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3.6. Fertilizing ability At the end of treatment period, fertilizing ability was lowest in pefloxacin-treated males, but the difference was not significant (Table 3). After cessation of treatment, fertilizing ability appeared to be recovering.
4. Discussion We consistently isolated E. coli from the foam. A reduction in E. coli counts was recorded in the foam even 2 h after the first treatment (data not shown). It was reported that fluoroquinolones penetrate very fast into the bacteria [23], which may be responsible for inhibition of DNA gyrase activity, an essential enzyme involved in DNA replication of bacteria [16,17]. Fluoroquinolones, therefore, disrupt DNA function, leading to death of the bacteria, consistent with the decreased number of bacterial counts in birds that received antibiotic treatment. That the decrease in E. coli counts occurred at the same time as reduced foam production in antibiotic-treated birds suggested that the bacteria have a role in foam formation. Although this is the first report on the effect of fluoroquinolones on foam production, McFarland et al. [6] also hypothesized the presence of bacteria in cloacal gland of Japanese quail and postulated their role in foam synthesis. More studies are needed to clearly elucidate the role of bacteria in foam production. The decline in fertilizing ability, body weight and area of cloacal gland was more prominent in the pefloxacin-treated birds compared to the other groups. Recently, we reported that the area of cloacal gland was positively correlated with plasma testosterone concentration, body weight and fertilizing ability of male Japanese quail [5]. Further, regression of cloacal gland in male Japanese quail by cyproterone acetate administration suggested that the cloacal gland is androgen-dependent and involved in maintenance of fertility [24]. Therefore, a reduction in cloacal gland area in the pefloxacin group may be partly responsible for decline in fertility and body weight in this group. Alternatively, the drop in fertility in the present study might be due to a toxic effect of the antibiotics on spermatozoa as well as spermatogonia and a secondary effect on steroid hormone negative feedback via the testes on the hypothalamus. That pefloxacin caused greater suppression of cloacal gland area and a trend towards greater suppression of body weight and fertilizing ability may be due to the shorter half-life of ciprofloxacin compared to other fluoroquinolones [25]. In that regard, pefloxacin concentrations in the cloacal gland and other tissues (breast muscle, kidney) at different time intervals after treatment (1, 5 and 10 days were several-fold higher than ciprofloxacin concentrations (unpublished observations). In conclusion, E. coli were present in the foam of the cloacal gland of Japanese quail and may have a role in foam production.
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