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Intraglandular injection of antibiotics for the treatment of vesicular adenitis in bulls
Animal Reproduction Science 104 (2008) 201–211
Intraglandular injection of antibiotics for the treatment of vesicular adenitis in bulls Marcelo F. Mart´ınez, Andres A. Arteaga, Albert D. Barth ∗ Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Dr., Saskatoon, Saskatchewan, Canada S7N 5B4 Received 1 September 2006; accepted 27 February 2007 Available online 6 March 2007
Abstract Two experiments were designed to determine the efficacy of intraglandular antibiotic treatment in beef bulls. Experiment 1 was designed to evaluate the glandular tissue reaction to intraglandular antibiotic treatment. Experiment 2 was conducted to determine the efficacy intraglandular injection of antibiotics for the treatment of naturally occurring cases of vesicular adenitis. Healthy beef bulls (n = 15), 2 and 3 years of age, were randomly allocated to three equal treatment groups to receive 10% of the daily recommended parenteral dose of penicillin, ceftiofur, or oxytetracycline in a volume of 6 mL injected directly into one of the vesicular glands. Ultrasonography was performed before, immediately after, and at 24, 48 and 168 h after intraglandular injection. The size and hardness of vesicular glands injected with oxytetracycline was greater (P < 0.01) than those injected with ceftiofur. Ultrasonographic pixel intensity increased (P < 0.01) after treatment with antibiotics, especially after treatment with oxytetracycline or penicillin. In Experiment 2, yearling beef bulls with clinical vesicular adenitis (n = 14) were referred to the Western College of Veterinary Medicine for treatment. Eight bulls had unilateral and six had bilateral vesicular adenitis. The most common isolate was Arcanobacterium pyogenes. Corynebacterium pseudotuberculosis was isolated from one bull. Bulls were subjected to rectal palpation and ultrasonography of the vesicular glands, semen collection by electroejaculation, and intraglandular treatment with ceftiofur (n = 13) and if necessary, a second intraglandular treatment of penicillin (n = 6). One bull was treated only with an initial intraglandular injection of penicillin. Bulls were evaluated once a week over 6 weeks by palpation of the glands, and evaluation of semen. All bulls recovered from vesicular adenitis after 3–6 weeks. There was a difference in the amount of pus (P = 0.042), leukocytes (P < 0.001) and blood (P = 0.003) present in ejaculates from before treatment to 3 weeks after treatment. Pixel intensities in ultrasonographic images of healthy or affected vesicular glands, whether treated or untreated, did not change over time.
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Corresponding author. Tel.: +1 306 966 7151; fax: +1 306 966 7159. E-mail address: [email protected] (A.D. Barth).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.02.025
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Intraglandular injection of ceftiofur in yearling bulls via the ischiorectal fossa was effective for treating vesicular adenitis. © 2007 Elsevier B.V. All rights reserved. Keywords: Bulls—vesicular adenitis; Intraglandular antibiotic injection
1. Introduction Vesicular adenitis is an important cause of economic loss for producers of beef bulls. In most cases there are no overt clinical signs and the disease is first detected when bulls are submitted for breeding soundness evaluation (BSE). The prevalence of vesicular adenitis is highest in yearling bulls and occurs unilaterally or bilaterally in 0.85–10% of bulls (Cavalieri and Van Camp, 1997). A large variety of organisms have been isolated from cases of vesicular adenitis, however, in North America Arcanobacterium pyogenes and Histophilus somni are the most common isolates. The hypothetical pathogenesis of this disease includes different routes of infection such as hematogenous invasion from a previous nidis of infection (Dargatz et al., 1987), from rumenitis due to rations with a great amount of energy content (Cavalieri and Van Camp, 1997), or anomalies of the colliculus seminalis that could predispose to reflux of urine into the vesicular gland (Blom, 1979a,b). Occlusion of large ducts of the vesicular glands by proteinaceous material and epithelial debris causing distension of the glandular alveoli has also been suggested in the pathogenesis (Ball et al., 1968). The most common method of treatment of vesicular adenitis is with parenteral injection of antibiotics. Surgical removal of affected glands has been reported and there is evidence that some bulls recover spontaneously from vesiculitis (Larson, 1997). Parenteral antibiotic treatment has given variable results (Phillips, 1993). Earlier reports of intraglandular injections described techniques of bacterial or Mycoplasma spp. inoculation with an 18-gauge needle through the rectum wall (Blom and Ernø, 1967), ischiorectal fossa (Al-Aubaidi et al., 1972; La Faunce and McEntee, 1982), or gluteal muscles (Hoover, 1974). There have been anecdotal reports of intraglandular injections of sclerosing agents (Hoover, 1974); however, intraglandular use of antibiotics has not been reported. Experiment 1 was designed to evaluate the tissue reaction of healthy vesicular glands to intraglandular antibiotic injection and Experiment 2 was conducted to determine the efficacy intraglandular injection of antibiotics in bulls with vesicular adenitis. 2. Materials and methods 2.1. Experiment 1 Protocols for animal use in both experiments were approved by the University of Saskatchewan Animal Care and Use Committee. Hereford bulls (n = 25), aged 2–3 years and weighing 703.9 ± 24.8 kg, were maintained on alfalfa/grass hay with free access to water in outdoor pens at a University of Saskatchewan research farm. Bulls were randomly allocated to three treatment groups of seven bulls each to receive 10% of the daily recommended parental dose of penicillin G procaine (22,000 IU/kg IM; Pen G injection® , Citadel Animal Health, Cambridge, Ontario), ceftiofur (1 mg/kg kg IM; Excenell® , Pharmacia and Upjohn Sant´e Animale, Orangeville, Ontario), or oxytetracycline (10 mg/kg; Sigma–Aldrich Canada Ltd., Oakville, ON) in a volume of 6 mL made up with saline solution and injected directly into a vesicular gland.
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Oxytetracycline powder was suspended in saline and balanced to a pH of 7. A control group of four bulls received no treatment. The method of intraglandular injection of antibiotics was as follows: After the rectum was manually evacuated, the pararectal region was clipped and scrubbed with 1% povidone-iodine solution. Local analgesia was performed by caudal epidural injection of 6 mL of 2% lidocaine HCl (Bimeda-MTC Animal Health Inc., Cambridge, Ontario) and by infiltration of the subcutaneous tissue of the ischiorectal fossa with 6 mL of 2% lidocaine. A 1 cm stab incision was made in the skin of the ischiorectal fossa. A custom-made, rigid, blunt, 4 mm outside-diameter, needle guide was pushed through the incision and passed pararectally up to the vesicular gland. The caudal reflection of the peritoneum was bluntly perforated with a short jab. The vesicular gland was fixed by one hand in the rectum while the needle guide was manipulated with the opposite hand. A 44 cm long 18-gauge needle containing antibiotic (to avoid injecting air into the gland) and connected to a syringe with antibiotic was directed through the needle guide into the gland. Needle-placement within the gland was verified by manually moving the gland and observing the corresponding needle movement. Changes in gland tissue echo texture immediately after antibiotic injection could also be used to verify antibiotic deposition within the gland. The antibiotic was slowly injected into the gland. Ultrasonography of treated and untreated glands was performed before, immediately after, and at 24, 48 and 168 h after antibiotic injection. 2.2. Experiment 2 Untreated beef bulls (n = 14) with clinical vesicular adenitis (enlarged, hardened glands and curds of pus present in semen samples) were referred to the Western College of Veterinary Medicine, University of Saskatchewan, for treatment. They were maintained under similar conditions as bulls of Experiment 1. Semen was collected in a sterile manner by electroejaculation for bacterial culture at the initial evaluation. After penile protrusion, the glans penis was grasped with a sterile surgical sponge and the tip of the penis was swabbed with betadine and dried with a sterile sponge. A volume of semen was directed into a sterile vial held horizontally near the penis. Semen was swabbed on sheep blood agar and MacConkey agar. All plates were incubated at 37 ◦ C in an atmosphere of 7% CO2 . The plates were examined for bacterial growth once a day for 72 h. If a plate had growth of more than 10 bacterial colonies, subcultures were prepared for further identification. An impression smear was also prepared from each swab and gram stained. Biochemical characterization and antimicrobial sensitivity tests for bacteria isolated in the study were performed following the National Committee for Clinical Laboratory Standards M31-A recommendations (NCCLS, 1997). Bulls were subjected to transrectal palpation and ultrasonography of the vesicular glands and semen collection by electroejaculation before treatment and every 4 days (11 times) after treatment. Ceftiofur was injected into affected glands as the initial treatment in 13 bulls and, if there was no evidence of recovery within 3 weeks, a second intraglandular treatment with penicillin (n = 6) was conducted. One bull was treated only once with an initial intraglandular injection of penicillin. Three bulls (bulls 1, 10, and 12, Table 1) that did not recover from vesiculitis after two intraglandular antibiotic injections received three doses of tilmicosin subcutaneously (10 mg/kg; Micotil® , Provel/Elanco Animal Health, Guelph, Ontario) 48 h apart. In these bulls, additional semen samples were taken every 4 days for as long as 10 weeks from the initiation of treatment. Recovery from vesicular adenitis was considered to have occurred when semen samples no longer contained any visual evidence of pus or blood and the average number of leukocytes was < 1 per five microscope fields in at least 15 fields observed at a magnification of 1000×.
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Table 1 Bacteria isolated from yearling beef bulls affected with vesicular adenitis Bull no.
Main isolate
Concomitant isolates
1 2 3 4 5 6 7 8 9 10 11 12 13 14
A. pyogenes Acinetobacter sp. Corynebacterium sp. C. pseudotuberculosis Acinetobacter sp. Streptococcus sp. A. pyogenes G (-) rods A. pyogenes No isolates No isolates No isolates G (-) rods A. pyogenes
Corynebacterium sp., Pasteurela sp. Pasteurella sp. Corynebacterium sp.
Corynebacterium sp., G (-) rods G (-) rods
Semen evaluation was done according to the method of the Western Canadian Association of Bovine Practitioners (Barth, 2000). Semen density was considered very good (VG) in semen with a grainy appearance with approximately 0.75–1 billion sperm per mL, good (G) for opaque milk-like semen with approximately 0.40–0.75 billion sperm per mL, fair (F) for skim milklike semen with approximately 0.25–0.40 billion sperm per mL, and poor (P) for translucent or watery semen with less than 0.25 billion sperm per mL. Gross motility was considered VG for semen with vigorous swirling, G for semen with slower swirling, F for semen with a generalized oscillation and P for semen with sporadic movement. For data analysis, density and gross motility classifications were replaced by scores: 1 = VG, 2 = G, 3 = F, 4 = P. The presence of blood, pus and leukocytes in the semen, and vesicular gland hardness were also scored from 0 to 4. For blood 0 = absence, 1 = slight tinge, 2 = pink, 3 = red, 4 = dark red ± clots. For pus, 0 = absence, 1 = a few 1 mm diameter flakes and 2 = a few 2 mm diameter curds, at the bottom of the semen column after a settling period of 5 min, 3 = up to a half mL and 4 = >a half mL of pus at the bottom of the semen column immediately after semen collection. For detection of leukocytes, semen was allowed to sit for 5 min and then semen was sampled from the bottom of the semen column for preparation of an eosin–nigrosin stained semen smear. A score of 0 = absence, 1 = < 2 leukocytes per 1000× field on microscopy, 2 = between 2 and 5 leukocytes per field, 3 = 5 to 10 leukocytes per field, 4 = more than 10 leukocytes per field. Sperm were considered to be alive if they did not take up eosin in the stained semen smears. The hardness of the vesicular glands was subjectively scored by transrectal palpation as 1 = consistency of a normal gland, 2 = slightly increased firmness, 3 = moderately hard, 4 = hard. 2.3. Ultrasonographic imaging In Experiment 1, transrectal ultrasonographic examinations were performed immediately before, immediately after and at 24, 72 and 168 h after intraglandular antibiotic treatment to monitor changes in glandular echotexture and changes in volume. A crude measure of volume was determined by length × width × depth measurements through the central region of glands. A real-time, B-mode scanner (Model SSD-900; Aloka, Tokyo, Japan) equipped with a 7.5 MHz
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Fig. 1. Image captured and analyzed by the built-in program at the time of an ultrasonogram. There is a histogram of the pixel intensities for each of three 1 cm2 areas depicted on the screen. On the right side of the image, the total number of pixels (T), the intensity level (L) that occurred most frequently, the number of pixels (M) corresponding to the intensity level that most frequently occurred, the mean intensity level (MN), the standard deviation of the intensity distribution (S.D.) in each specified area are shown. These data were used in the statistical analysis.
linear-array transducer was used. The ultrasonographic scanner had a built-in program that allowed for the construction of a histogram that indicated the intensity distribution of image pixels within a randomly selected 1 cm2 area (Fig. 1). The horizontal axis of the frequency distribution indicated intensity levels between 0 and 63, and the vertical axis indicated the percentage of pixels of each intensity level. The data provided by the built-in program included T, the total number of pixels in an arbitrary area or a fixed area; L, intensity level that occurred most frequently in the specified area; M, number of pixels corresponding to the intensity level that most frequently occurred in the specified area; MN, mean intensity level in the specified area; S.D., standard deviation of the intensity distribution in the specified area. A mean high intensity pixel value was also calculated based on L multiplied by its frequency M. Percentage of MN and S.D. were also analyzed using the ultrasonogrphic image at time 0 (before treatment) as the reference control equal to 100%. 2.4. Statistical analysis In Experiment 1, ultrasonographic vesicular gland measurements (length, height, depth) were used to determine volume of the glands. Gland volumes were analyzed by the Kruskal–Wallis non-parametric test and mean ranks of volume between groups were further analyzed by the Mann–Whitney test. Homogeneity of variances of gland volume was analyzed by Levene’s test. In Experiment 2, Pearson’s correlation coefficients were calculated for semen characteristics (density, gross motility, individual motility, percentage of normal sperm, presence of blood, pus, or leukocytes). Changes in semen traits (gross motility, individual motility, percentage of normal sperm, percentage of head defects and percentage of midpiece defects) before and after treatments were compared using the Kruskal–Wallis non-parametric test. Scores of consistency of the
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vesicular glands and the presence of blood, pus or leukocytes among semen samples were also compared by the Kruskal–Wallis non-parametric test. Mean ranks between groups were further compared by the Mann–Whitney test. In Experiment 1, the mean value of high intensity pixels, MN, percentage of MN, and S.D. of the intensity distribution were analyzed by mixed procedure with first order autoregressive covariance (Littell et al., 1998). Main effects in data points were further compared by LSD test. In Experiment 2, bulls were treated in the affected gland(s), i.e., bulls with unilateral vesiculitis received treatment in only one gland. Therefore, pixel data from healthy, non-treated glands of bulls with unilateral vesiculitis were compared with those from affected glands immediately before and 7 days after treatment. MN and S.D. of pixel distribution were analyzed by analysis of variance in a 2 × 2 factorial design (Status: affected or healthy gland; time: before and 7 days after treatment). Data and graph processing were performed with Microsoft Office Excel and statistical analyses were conducted with SPSS for Windows (SPSS v. 11.5, SPSS Inc. 2002, USA). 3. Results 3.1. Experiment 1 Seven days after intraglandular injection of antibiotic, the volume of the vesicular glands treated with oxytetracycline was greater (P < 0.01) than in those that received ceftiofur, or were not treated, whereas the volume of glands treated with penicillin was intermediate (P < 0.09; Fig. 2). However, one bull in the ceftiofur group had similar glandular size to those in oxytetracycline or penicillin groups. Variability of the glandular volume was greater (P < 0.05) in glands treated with penicillin and oxytetracycline than in those treated with ceftiofur, or not treated. The latter two groups had a similar volume (P < 0.2).
Fig. 2. Box-plot of the volume of vesicular glands 7 days after intraglandular treatment with ceftiofur, oxytetracycline or penicillin in healthy beef bulls. The control group is represented by those glands that did not receive treatment. + and − represent mean and median volume, respectively, while × represents the outliers according to SPSS program. Boxes are composed by the first and third quartiles while the top and bottom points of the central lines represent the maximum and minimum values. Letters a and b mean ranks with different letters differ (P < 0.01).
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Fig. 3. Percentage change in the mean pixel intensity from the pretreatment values after intravesicular gland antibiotic injections in Experiment 1.
3.2. Ultrasonography of the vesicular glands The mean (±S.E.M.) value of high intensity pixels over 168 h after intraglandular injection of antibiotics tended to be influenced by treatment (P = 0.15), where there was an effect of time (P = 0.006), but no effect of treatment by time interaction (P = 0.52). The mean intensity level of pixel value changed over time (P < 0.001), but was not affected by the type of antibiotic treatment (P = 0.23) or the interaction of treatment and time (P = 0.47). However, there was an effect of treatment (P = 0.032), and time (P < 0.001), but no treatment-by-time interaction (P = 0.40) on the percentage of MN. In general, the percentage of change in the mean pixel value was greater (P < 0.05) in the bulls treated with penicillin or oxytetracycline than that in bulls treated with ceftiofur. A change (P = 0.05) in the percentage of MN occurred immediately after injection of the antibiotic (Fig. 3). There was a tendency for an effect of antibiotic (P = 0.07) and an effect of time (P < 0.001), but there was no effect of antibiotic-by-time interaction (P = 0.69) on the percentage of S.D. of the intensity distribution of pixels. 3.3. Experiment 2 There were eight bulls with unilateral (left, n = 3; right, n = 5) and six bulls with bilateral vesicular adenitis. Data for types of bacteria isolated from the different bulls are shown in Table 1. All bacteria were sensitive to ceftiofur, penicillin and tilmicosin. All bulls recovered from the vesicular adenitis after one or more treatments. There was a significant difference among ejaculates in pus (P = 0.042), leukocytes (P < 0.001) and blood (P = 0.003) (Fig. 4). The mean change in consistency score of the glands estimated by rectal palpation remained unremarkable from the first to the last examination (P = 0.63); however, in three bulls the consistency score decreased from 4 to 1 by the time of the last examination. A high correlation was found between semen density and gross motility (r = 0.51; P < 0.0001), gross and individual motility (r = 0.68; P < 0.001), percentage of live sperm and individual motility (r = 0.64; P < 0.0001), and between individual motility and percentage of normal sperm (r = 0.46;
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Fig. 4. Mean scores representing the consistency of the vesicular glands and the presence of pus, leukocytes and blood in the semen samples obtained before and after intraglandular antibiotic injection from all bulls affected with vesicular adenitis. Semen samples were collected every 4 days. Letters a and b means of scores with different superscripts differ (P < 0.05).
P < 0.0001). There was a significant negative correlation between the presence of leukocytes in semen and percentage of live sperm (r = −0.24; P < 0.02), and between leukocytes and the percentage of midpiece defects (r = −0.24; P = 0.02). There was no difference from the first to last semen samples, before and after treatments, in sperm morphology (P = 0.24); however, motility improved when pus was no longer present in semen (gross motility, P = 0.05; individual motility, P < 0.08). When inflamed and healthy vesicular glands were compared by ultrasonography before and 1 wk after treatment, there was no effect of gland health status, time or health status by time interaction on the MN (P = 0.3, P = 0.46, and P = 0.64, respectively) or S.D. (P = 0.16, P = 0.77, and P = 0.20, respectively) of pixel distribution. 4. Discussion This is the first report of studies on intraglandular injections of antibiotics in beef bulls using the ischiorectal fossa as a route of administration and the first report of ultrasonographic pixel characterization of inflamed and healthy vesicular glands with or without intraglandular antibiotic treatment. Previous studies have focused mainly on the description of pathogenesis, pathological findings and diagnosis rather than treatment of vesicular adenitis (Dargatz et al., 1987; Galloway, 1964; Blom, 1979a,b). The pararectal approach for gland injection allowed good control of the positioning of the needle for gland injection and no appreciable adverse reaction was found after this procedure in the animals’ behavior or by rectal palpation and ultrasonography. In the present study, the
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type of intraglandularly injected antibiotic influenced the volumetric size of vesicular glands. A greater glandular volume was observed in those treated with oxytetracycline chlorhydrate or penicillin than in those not treated or treated with ceftiofur. It appeared that ceftiofur induced less inflammation and might be the more suitable antibiotic for intraglandular treatment. Adverse consequences of greater inflammation could include the development of adhesions between the gland and surrounding tissue as has been observed previously when iodine solution was used to treat the condition (Hoover, 1974). The lesser degree of inflammation provoked by ceftiofur is most likely beneficial clinically and on welfare grounds. The proposed differences in tissue reaction induced by the different antibiotics were also supported by the differences in pixel MN values that were observed over time. Ultrasonography has been used for pixel analysis of testicular tissue in bulls (Aravindakshan et al., 2000; Brito et al., 2003) and testes and accessory glands of growing healthy bull calves (Chandolia et al., 1997), but the use of ultrasonography to study the effect of an antibiotic injection into the vesicular glands has not been previously reported. Simple visual observations of ultrasonograms revealed a dramatic difference among treatments in gland size and echogenicity. Computer analysis of pixel intensity distribution in ultrasonograms confirmed the visual observations of increased echogenicity of vesicular glands after injection with antibiotics. A remarkable change was noticed in the mean pixel intensity of ultrasonograms taken immediately after antibiotic injection. The mean pixel intensity value MN was greater in glands treated with oxytetracycline and penicillin compared to untreated glands or those treated with ceftiofur. Each antibiotic solution might have had differences in echogenicity from other antibiotics and this characteristic might have affected the tissue echogenicity immediately after injection. However, a direct measurement of the pixel intensities of antibiotic solutions was not conducted. Oxytetracycline- and penicillin-treated glands continued to have a higher percentage of MN compared to ceftiofur in ultrasonographic scans at 24, 72 and 168 h after treatment. This continued increase in pixel intensity concurs with findings on rectal palpation of glands in the post-treatment period when antibiotic-treated glands felt enlarged and firmer in texture. The pixel MN returned to pre-treatment level values 7 days after treatment with ceftiofur, whereas, the pixel MN for oxytetracline- and penicillin-treated glands remained elevated. This would indicate that ceftiofur was less irritating to the tissue than the other antibiotics. In bulls with vesiculitis, there was no change in the pixel intensity in glands from pretreatment scans to ultrasound examinations 7 days after treatment. Injection of antibiotic into the glands resulted in the presence of blood in semen samples regardless of the type of antibiotic used. Presumably bleeding occurred due to tearing of tissue at the site of antibiotic deposition. The presence of blood in semen samples declined to undetectable levels after 28 days and while tissue hemorrhage is undesirable in the short term, there appeared to be no lasting adverse effect. Intraglandular treatment did not adversely affect semen quality as sperm morphology did not change over the period of treatment and there was a beneficial effect on sperm viability after pus was no longer present in semen. The antibiotic distribution throughout the vesicular gland after direct injection might be a concern. Even though glands were always injected approximately in the middle, immediate ultrasound scans showed that the antibiotic did not reach some peripheral areas of the glands. This could only be assessed in the oxytetracycline- and penicillin-treated groups because these antibiotics caused greater gland tissue echogenicity than ceftiofur. In addition, if the tip of the needle was placed in or near a large glandular duct there could be loss of antibiotic from the injection site through a duct. Another important aspect in the glandular distribution of the antibiotic is the nature of the antibiotic. The third generation of cephalosporins, to which ceftiofur belongs, and procaine peni-
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cillin G have an extensive distribution in extracellular fluid, but the passage across membranes or other physiological barriers is poor (Prescott, 2002a,b). Penetration of physiological barriers by penicillin is increased in inflammatory processes (Prescott, 2002b). This may have been the cause of success in the intraglandular treatment of infected glands in a bull in Experiment 2. A. pyogenes was the most common kind of bacterium isolated from the infected bulls and in three cases was associated with a concomitant bacterium. It has also been the most common isolate reported in the literature (Cavalieri and Van Camp, 1997; Carroll et al., 1968; Phillips, 1993; Mickelsen et al., 1994; Carson, 2000). Blom and Christensen (1965) reported that A. pyogenes was cultured in 27.4% of 6211 semen samples examined while Escherichia coli was isolated from 20% of semen samples. In the present study, E. coli was isolated in three bulls. A particularly interesting finding was the isolation from one bull of Corynebacterium pseudotuberculosis which has not been reported previously in cases of vesicular adenitis. This bull received only one treatment with ceftiofur and in the subsequent semen collections showed a significant improvement. There were also other Corynebacteria isolated from semen samples. In most cases, Corynebacterium sp. were cultured in association with other bacteria such as Pasteurella sp. and A. pyogenes. A. pyogenes was found in 37.7% of 106 preputial washings while Corynebacterium renale and other Corynebacterium sp. were found in 53.8% (Galloway, 1964). These bacteria were also cultured from vesicular glands obtained post-mortem. This led to speculation that bacteria that usually colonize the prepuce and penis may cause vesicular adenitis using the hematogenous route of infection through small lacerations in the glans penis when bulls mount each other. Isolation of Streptococcus sp. in a small percentage of bulls with vesiculitis has been reported previously (Ball et al., 1968), and was seen in one of the 14 bulls in the present study. In other published cases of vesicular adenitis, at times no bacteria could be isolated. In the present study, no bacteria were cultured from three bulls from the same farm. This might have been due to unreported antibiotic treatment of the bulls before they were submitted for the present experiment. Due to a limited availability of clinical cases and limited funding for the transport and care of bulls, an untreated control group was not used. This is a shortcoming of Experiment 2 because an untreated control group was necessary to verify that the treatments used were more beneficial than no treatment. Further studies are needed to verify the conclusions of the present study. 5. Conclusion The present experiments indicated that intraglandular injection of ceftiofur or penicillin in beef bulls via the ischiorectal fossa appeared to be safe and effective in the treatment of vesicular adenitis. Ultrasonography can be used to evaluate the characteristics of the glands prior to and after intraglandular antibiotic treatment. Evaluation of vesicular glands by rectal palpation is also useful in monitoring the effect of treatments, but semen collection is likely the most useful method to evaluate the progress of recovery from vesiculitis. The present study also gave information about the etiology of vesiculitis of western Canadian bulls and it constitutes the first report of isolation of C. pseudotuberculosis from a case of vesicular adenitis in a bull. References Al-Aubaidi, J.M., McEntee, K., Lein, D.H., Roberts, S.J., 1972. Bovine seminal vesiculitis and epididymitis caused by Mycoplasma bovigenitalium. Cornell Vet. 59, 589–596. Aravindakshan, J.P., Honaramooz, A., Bartlewski, P.M., Beard, A.P., Pierson, R.R., Rawlings, N.C., 2000. Gonadotrophin secretion in prepubertal bull calves born in spring and autumn. J. Reprod. Fertil. 120, 159–167.
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Ball, L., Young, S., Carroll, E.J., 1968. Seminal vesiculitis syndrome, lesions in genital organs of young bulls. Am. J. Vet. Res. 29, 1173–1184. Barth, A.D., 2000. Bull Breeding Soundness Evaluation. The Western Canadian Association of Bovine Practitioners, Calgary. Blom, E., 1979a. Studies on seminal vesiculitis in the bull. I. Semen examination methods and post mortem findings. Nord. Vet. Med. 31, 193–205. Blom, E., 1979b. Studies on seminal vesiculitis in the bull. II. Malformation of the pelvic genital organs as a possible predisposing factor in the pathogenesis of seminal vesiculitis. Nord. Vet. Med. 31, 241–250. Blom, E., Christensen, N.O., 1965. Seminal vesiculitis in the bull caused by Corynebacterium pyogenes. Nord. Vet. Med. 17, 435–445. Blom, E., Ernø, H., 1967. Mycoplasmosis, Infections of the genital organs of bulls. Acta Vet. Scand. 8, 186–188. Brito, L.F.C., Silva, A.E.D.F., Barbosa, R.T., Unanian, M.M., Kastelic, J.P., 2003. Effects of scrotal insulation on sperm production, semen quality, and testicular echotexture in Bos indicus and Bos indicus-Bos taurus bulls. Anim. Reprod. Sci. 79, 1–15. Carroll, E.J., Ball, L., Young, S., 1968. Seminal vesiculitis in young beef bulls. J. Am. Vet. Med. Assoc. 152, 1749–1757. Carson, R.L., 2000. Diseases of the male internal genitalia. In: Smith, H. (Ed.), Current Veterinary Therapy, Food Animal Practice. W.B. Saunders Co. publishers, Philadelphia, PA. Cavalieri, J., Van Camp, S.D., 1997. Bovine seminal vesiculitis. A review and update. Vet. Clin. North Am. Food Anim. Pract. 13, 233–241. Chandolia, R.K., Honaramooz, A., Omeke, B.C., Pierson, R., Beard, A.P., Rawlings, N.C., 1997. Assessment of development of the testes and accessory glands by ultrasonography in bull calves and associated endocrine changes. Theriogenology 48, 119–132. Dargatz, D.A., Mortimer, R.G., Ball, L., 1987. Vesicular adenitis of bulls, a review. Theriogenology 28, 513–521. Galloway, D.B., 1964. A study of bulls with the clinical signs of seminal vesiculitis, clinical, bacteriological, and pathological aspects. Acta Vet. Scand. 5 (Suppl. 2), 1–222. Hoover, T.R., 1974. Bacterial seminal vesiculitis in bulls. In: Proceedings of the Society for Theriogenology, Mobile, AL, pp. 92–98. La Faunce, N.A., McEntee, K., 1982. Experimental Mycoplasma bovis seminal vesiculitis in the bull. Cornell Vet. 72, 150–167. Larson, L.L., 1997. Diagnosing and controlling seminal vesiculitis in bulls. Vet. Med. 92, 1073–1078. Littell, R.C., Henry, P.R., Ammerman, C.B., 1998. Statistical analysis of repeated measures data using SAS procedures. J. Anim. Sci. 76, 1216–1231. Mickelsen, W.D., Weber, J.A., Memon, M.A., 1994. Use of transrectal ultrasound for the detection of seminal vesiculitis in a bull. Vet. Rec. 135, 154–155. National Committee for Clinical Laboratory Standards, 1997. Performance Standards for Antimicrobial Disk Susceptibility Tests, 6th ed. National Committee for Clinical Laboratory Standards, Wayne, Pennsylvania, USA, Approved standard M2-A6 (M100-S7). Phillips, P.E., 1993. Seminal vesiculitis, new strategies for an old problem. In: Proceedings of the Society for Theriogenology Jacksonville, Florida, USA, pp. 59–66. Prescott, J.F., 2002a. Beta-lactam antibiotics, cephalosporins and cephamycins. In: Prescott, J.F., Baggott, J.D., Walker, R.D. (Eds.), Antimicrobial Therapy in Veterinary Medicine, 3rd ed. Iowa State University Press, Ames, USA, pp. 134–159. Prescott, J.F., 2002b. Beta-lactam antibiotics, penam penicillins. In: Prescott, J.F., Baggott, J.D., Walker, R.D. (Eds.), Antimicrobial Therapy in Veterinary Medicine, 3rd ed. Iowa State University Press, Ames, USA, pp. 105–133.
Intraglandular injection of antibiotics for the treatment of vesicular adenitis in bulls Marcelo F. Mart´ınez, Andres A. Arteaga, Albert D. Barth ∗ Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Dr., Saskatoon, Saskatchewan, Canada S7N 5B4 Received 1 September 2006; accepted 27 February 2007 Available online 6 March 2007
Abstract Two experiments were designed to determine the efficacy of intraglandular antibiotic treatment in beef bulls. Experiment 1 was designed to evaluate the glandular tissue reaction to intraglandular antibiotic treatment. Experiment 2 was conducted to determine the efficacy intraglandular injection of antibiotics for the treatment of naturally occurring cases of vesicular adenitis. Healthy beef bulls (n = 15), 2 and 3 years of age, were randomly allocated to three equal treatment groups to receive 10% of the daily recommended parenteral dose of penicillin, ceftiofur, or oxytetracycline in a volume of 6 mL injected directly into one of the vesicular glands. Ultrasonography was performed before, immediately after, and at 24, 48 and 168 h after intraglandular injection. The size and hardness of vesicular glands injected with oxytetracycline was greater (P < 0.01) than those injected with ceftiofur. Ultrasonographic pixel intensity increased (P < 0.01) after treatment with antibiotics, especially after treatment with oxytetracycline or penicillin. In Experiment 2, yearling beef bulls with clinical vesicular adenitis (n = 14) were referred to the Western College of Veterinary Medicine for treatment. Eight bulls had unilateral and six had bilateral vesicular adenitis. The most common isolate was Arcanobacterium pyogenes. Corynebacterium pseudotuberculosis was isolated from one bull. Bulls were subjected to rectal palpation and ultrasonography of the vesicular glands, semen collection by electroejaculation, and intraglandular treatment with ceftiofur (n = 13) and if necessary, a second intraglandular treatment of penicillin (n = 6). One bull was treated only with an initial intraglandular injection of penicillin. Bulls were evaluated once a week over 6 weeks by palpation of the glands, and evaluation of semen. All bulls recovered from vesicular adenitis after 3–6 weeks. There was a difference in the amount of pus (P = 0.042), leukocytes (P < 0.001) and blood (P = 0.003) present in ejaculates from before treatment to 3 weeks after treatment. Pixel intensities in ultrasonographic images of healthy or affected vesicular glands, whether treated or untreated, did not change over time.
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Corresponding author. Tel.: +1 306 966 7151; fax: +1 306 966 7159. E-mail address: [email protected] (A.D. Barth).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.02.025
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Intraglandular injection of ceftiofur in yearling bulls via the ischiorectal fossa was effective for treating vesicular adenitis. © 2007 Elsevier B.V. All rights reserved. Keywords: Bulls—vesicular adenitis; Intraglandular antibiotic injection
1. Introduction Vesicular adenitis is an important cause of economic loss for producers of beef bulls. In most cases there are no overt clinical signs and the disease is first detected when bulls are submitted for breeding soundness evaluation (BSE). The prevalence of vesicular adenitis is highest in yearling bulls and occurs unilaterally or bilaterally in 0.85–10% of bulls (Cavalieri and Van Camp, 1997). A large variety of organisms have been isolated from cases of vesicular adenitis, however, in North America Arcanobacterium pyogenes and Histophilus somni are the most common isolates. The hypothetical pathogenesis of this disease includes different routes of infection such as hematogenous invasion from a previous nidis of infection (Dargatz et al., 1987), from rumenitis due to rations with a great amount of energy content (Cavalieri and Van Camp, 1997), or anomalies of the colliculus seminalis that could predispose to reflux of urine into the vesicular gland (Blom, 1979a,b). Occlusion of large ducts of the vesicular glands by proteinaceous material and epithelial debris causing distension of the glandular alveoli has also been suggested in the pathogenesis (Ball et al., 1968). The most common method of treatment of vesicular adenitis is with parenteral injection of antibiotics. Surgical removal of affected glands has been reported and there is evidence that some bulls recover spontaneously from vesiculitis (Larson, 1997). Parenteral antibiotic treatment has given variable results (Phillips, 1993). Earlier reports of intraglandular injections described techniques of bacterial or Mycoplasma spp. inoculation with an 18-gauge needle through the rectum wall (Blom and Ernø, 1967), ischiorectal fossa (Al-Aubaidi et al., 1972; La Faunce and McEntee, 1982), or gluteal muscles (Hoover, 1974). There have been anecdotal reports of intraglandular injections of sclerosing agents (Hoover, 1974); however, intraglandular use of antibiotics has not been reported. Experiment 1 was designed to evaluate the tissue reaction of healthy vesicular glands to intraglandular antibiotic injection and Experiment 2 was conducted to determine the efficacy intraglandular injection of antibiotics in bulls with vesicular adenitis. 2. Materials and methods 2.1. Experiment 1 Protocols for animal use in both experiments were approved by the University of Saskatchewan Animal Care and Use Committee. Hereford bulls (n = 25), aged 2–3 years and weighing 703.9 ± 24.8 kg, were maintained on alfalfa/grass hay with free access to water in outdoor pens at a University of Saskatchewan research farm. Bulls were randomly allocated to three treatment groups of seven bulls each to receive 10% of the daily recommended parental dose of penicillin G procaine (22,000 IU/kg IM; Pen G injection® , Citadel Animal Health, Cambridge, Ontario), ceftiofur (1 mg/kg kg IM; Excenell® , Pharmacia and Upjohn Sant´e Animale, Orangeville, Ontario), or oxytetracycline (10 mg/kg; Sigma–Aldrich Canada Ltd., Oakville, ON) in a volume of 6 mL made up with saline solution and injected directly into a vesicular gland.
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Oxytetracycline powder was suspended in saline and balanced to a pH of 7. A control group of four bulls received no treatment. The method of intraglandular injection of antibiotics was as follows: After the rectum was manually evacuated, the pararectal region was clipped and scrubbed with 1% povidone-iodine solution. Local analgesia was performed by caudal epidural injection of 6 mL of 2% lidocaine HCl (Bimeda-MTC Animal Health Inc., Cambridge, Ontario) and by infiltration of the subcutaneous tissue of the ischiorectal fossa with 6 mL of 2% lidocaine. A 1 cm stab incision was made in the skin of the ischiorectal fossa. A custom-made, rigid, blunt, 4 mm outside-diameter, needle guide was pushed through the incision and passed pararectally up to the vesicular gland. The caudal reflection of the peritoneum was bluntly perforated with a short jab. The vesicular gland was fixed by one hand in the rectum while the needle guide was manipulated with the opposite hand. A 44 cm long 18-gauge needle containing antibiotic (to avoid injecting air into the gland) and connected to a syringe with antibiotic was directed through the needle guide into the gland. Needle-placement within the gland was verified by manually moving the gland and observing the corresponding needle movement. Changes in gland tissue echo texture immediately after antibiotic injection could also be used to verify antibiotic deposition within the gland. The antibiotic was slowly injected into the gland. Ultrasonography of treated and untreated glands was performed before, immediately after, and at 24, 48 and 168 h after antibiotic injection. 2.2. Experiment 2 Untreated beef bulls (n = 14) with clinical vesicular adenitis (enlarged, hardened glands and curds of pus present in semen samples) were referred to the Western College of Veterinary Medicine, University of Saskatchewan, for treatment. They were maintained under similar conditions as bulls of Experiment 1. Semen was collected in a sterile manner by electroejaculation for bacterial culture at the initial evaluation. After penile protrusion, the glans penis was grasped with a sterile surgical sponge and the tip of the penis was swabbed with betadine and dried with a sterile sponge. A volume of semen was directed into a sterile vial held horizontally near the penis. Semen was swabbed on sheep blood agar and MacConkey agar. All plates were incubated at 37 ◦ C in an atmosphere of 7% CO2 . The plates were examined for bacterial growth once a day for 72 h. If a plate had growth of more than 10 bacterial colonies, subcultures were prepared for further identification. An impression smear was also prepared from each swab and gram stained. Biochemical characterization and antimicrobial sensitivity tests for bacteria isolated in the study were performed following the National Committee for Clinical Laboratory Standards M31-A recommendations (NCCLS, 1997). Bulls were subjected to transrectal palpation and ultrasonography of the vesicular glands and semen collection by electroejaculation before treatment and every 4 days (11 times) after treatment. Ceftiofur was injected into affected glands as the initial treatment in 13 bulls and, if there was no evidence of recovery within 3 weeks, a second intraglandular treatment with penicillin (n = 6) was conducted. One bull was treated only once with an initial intraglandular injection of penicillin. Three bulls (bulls 1, 10, and 12, Table 1) that did not recover from vesiculitis after two intraglandular antibiotic injections received three doses of tilmicosin subcutaneously (10 mg/kg; Micotil® , Provel/Elanco Animal Health, Guelph, Ontario) 48 h apart. In these bulls, additional semen samples were taken every 4 days for as long as 10 weeks from the initiation of treatment. Recovery from vesicular adenitis was considered to have occurred when semen samples no longer contained any visual evidence of pus or blood and the average number of leukocytes was < 1 per five microscope fields in at least 15 fields observed at a magnification of 1000×.
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Table 1 Bacteria isolated from yearling beef bulls affected with vesicular adenitis Bull no.
Main isolate
Concomitant isolates
1 2 3 4 5 6 7 8 9 10 11 12 13 14
A. pyogenes Acinetobacter sp. Corynebacterium sp. C. pseudotuberculosis Acinetobacter sp. Streptococcus sp. A. pyogenes G (-) rods A. pyogenes No isolates No isolates No isolates G (-) rods A. pyogenes
Corynebacterium sp., Pasteurela sp. Pasteurella sp. Corynebacterium sp.
Corynebacterium sp., G (-) rods G (-) rods
Semen evaluation was done according to the method of the Western Canadian Association of Bovine Practitioners (Barth, 2000). Semen density was considered very good (VG) in semen with a grainy appearance with approximately 0.75–1 billion sperm per mL, good (G) for opaque milk-like semen with approximately 0.40–0.75 billion sperm per mL, fair (F) for skim milklike semen with approximately 0.25–0.40 billion sperm per mL, and poor (P) for translucent or watery semen with less than 0.25 billion sperm per mL. Gross motility was considered VG for semen with vigorous swirling, G for semen with slower swirling, F for semen with a generalized oscillation and P for semen with sporadic movement. For data analysis, density and gross motility classifications were replaced by scores: 1 = VG, 2 = G, 3 = F, 4 = P. The presence of blood, pus and leukocytes in the semen, and vesicular gland hardness were also scored from 0 to 4. For blood 0 = absence, 1 = slight tinge, 2 = pink, 3 = red, 4 = dark red ± clots. For pus, 0 = absence, 1 = a few 1 mm diameter flakes and 2 = a few 2 mm diameter curds, at the bottom of the semen column after a settling period of 5 min, 3 = up to a half mL and 4 = >a half mL of pus at the bottom of the semen column immediately after semen collection. For detection of leukocytes, semen was allowed to sit for 5 min and then semen was sampled from the bottom of the semen column for preparation of an eosin–nigrosin stained semen smear. A score of 0 = absence, 1 = < 2 leukocytes per 1000× field on microscopy, 2 = between 2 and 5 leukocytes per field, 3 = 5 to 10 leukocytes per field, 4 = more than 10 leukocytes per field. Sperm were considered to be alive if they did not take up eosin in the stained semen smears. The hardness of the vesicular glands was subjectively scored by transrectal palpation as 1 = consistency of a normal gland, 2 = slightly increased firmness, 3 = moderately hard, 4 = hard. 2.3. Ultrasonographic imaging In Experiment 1, transrectal ultrasonographic examinations were performed immediately before, immediately after and at 24, 72 and 168 h after intraglandular antibiotic treatment to monitor changes in glandular echotexture and changes in volume. A crude measure of volume was determined by length × width × depth measurements through the central region of glands. A real-time, B-mode scanner (Model SSD-900; Aloka, Tokyo, Japan) equipped with a 7.5 MHz
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Fig. 1. Image captured and analyzed by the built-in program at the time of an ultrasonogram. There is a histogram of the pixel intensities for each of three 1 cm2 areas depicted on the screen. On the right side of the image, the total number of pixels (T), the intensity level (L) that occurred most frequently, the number of pixels (M) corresponding to the intensity level that most frequently occurred, the mean intensity level (MN), the standard deviation of the intensity distribution (S.D.) in each specified area are shown. These data were used in the statistical analysis.
linear-array transducer was used. The ultrasonographic scanner had a built-in program that allowed for the construction of a histogram that indicated the intensity distribution of image pixels within a randomly selected 1 cm2 area (Fig. 1). The horizontal axis of the frequency distribution indicated intensity levels between 0 and 63, and the vertical axis indicated the percentage of pixels of each intensity level. The data provided by the built-in program included T, the total number of pixels in an arbitrary area or a fixed area; L, intensity level that occurred most frequently in the specified area; M, number of pixels corresponding to the intensity level that most frequently occurred in the specified area; MN, mean intensity level in the specified area; S.D., standard deviation of the intensity distribution in the specified area. A mean high intensity pixel value was also calculated based on L multiplied by its frequency M. Percentage of MN and S.D. were also analyzed using the ultrasonogrphic image at time 0 (before treatment) as the reference control equal to 100%. 2.4. Statistical analysis In Experiment 1, ultrasonographic vesicular gland measurements (length, height, depth) were used to determine volume of the glands. Gland volumes were analyzed by the Kruskal–Wallis non-parametric test and mean ranks of volume between groups were further analyzed by the Mann–Whitney test. Homogeneity of variances of gland volume was analyzed by Levene’s test. In Experiment 2, Pearson’s correlation coefficients were calculated for semen characteristics (density, gross motility, individual motility, percentage of normal sperm, presence of blood, pus, or leukocytes). Changes in semen traits (gross motility, individual motility, percentage of normal sperm, percentage of head defects and percentage of midpiece defects) before and after treatments were compared using the Kruskal–Wallis non-parametric test. Scores of consistency of the
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vesicular glands and the presence of blood, pus or leukocytes among semen samples were also compared by the Kruskal–Wallis non-parametric test. Mean ranks between groups were further compared by the Mann–Whitney test. In Experiment 1, the mean value of high intensity pixels, MN, percentage of MN, and S.D. of the intensity distribution were analyzed by mixed procedure with first order autoregressive covariance (Littell et al., 1998). Main effects in data points were further compared by LSD test. In Experiment 2, bulls were treated in the affected gland(s), i.e., bulls with unilateral vesiculitis received treatment in only one gland. Therefore, pixel data from healthy, non-treated glands of bulls with unilateral vesiculitis were compared with those from affected glands immediately before and 7 days after treatment. MN and S.D. of pixel distribution were analyzed by analysis of variance in a 2 × 2 factorial design (Status: affected or healthy gland; time: before and 7 days after treatment). Data and graph processing were performed with Microsoft Office Excel and statistical analyses were conducted with SPSS for Windows (SPSS v. 11.5, SPSS Inc. 2002, USA). 3. Results 3.1. Experiment 1 Seven days after intraglandular injection of antibiotic, the volume of the vesicular glands treated with oxytetracycline was greater (P < 0.01) than in those that received ceftiofur, or were not treated, whereas the volume of glands treated with penicillin was intermediate (P < 0.09; Fig. 2). However, one bull in the ceftiofur group had similar glandular size to those in oxytetracycline or penicillin groups. Variability of the glandular volume was greater (P < 0.05) in glands treated with penicillin and oxytetracycline than in those treated with ceftiofur, or not treated. The latter two groups had a similar volume (P < 0.2).
Fig. 2. Box-plot of the volume of vesicular glands 7 days after intraglandular treatment with ceftiofur, oxytetracycline or penicillin in healthy beef bulls. The control group is represented by those glands that did not receive treatment. + and − represent mean and median volume, respectively, while × represents the outliers according to SPSS program. Boxes are composed by the first and third quartiles while the top and bottom points of the central lines represent the maximum and minimum values. Letters a and b mean ranks with different letters differ (P < 0.01).
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Fig. 3. Percentage change in the mean pixel intensity from the pretreatment values after intravesicular gland antibiotic injections in Experiment 1.
3.2. Ultrasonography of the vesicular glands The mean (±S.E.M.) value of high intensity pixels over 168 h after intraglandular injection of antibiotics tended to be influenced by treatment (P = 0.15), where there was an effect of time (P = 0.006), but no effect of treatment by time interaction (P = 0.52). The mean intensity level of pixel value changed over time (P < 0.001), but was not affected by the type of antibiotic treatment (P = 0.23) or the interaction of treatment and time (P = 0.47). However, there was an effect of treatment (P = 0.032), and time (P < 0.001), but no treatment-by-time interaction (P = 0.40) on the percentage of MN. In general, the percentage of change in the mean pixel value was greater (P < 0.05) in the bulls treated with penicillin or oxytetracycline than that in bulls treated with ceftiofur. A change (P = 0.05) in the percentage of MN occurred immediately after injection of the antibiotic (Fig. 3). There was a tendency for an effect of antibiotic (P = 0.07) and an effect of time (P < 0.001), but there was no effect of antibiotic-by-time interaction (P = 0.69) on the percentage of S.D. of the intensity distribution of pixels. 3.3. Experiment 2 There were eight bulls with unilateral (left, n = 3; right, n = 5) and six bulls with bilateral vesicular adenitis. Data for types of bacteria isolated from the different bulls are shown in Table 1. All bacteria were sensitive to ceftiofur, penicillin and tilmicosin. All bulls recovered from the vesicular adenitis after one or more treatments. There was a significant difference among ejaculates in pus (P = 0.042), leukocytes (P < 0.001) and blood (P = 0.003) (Fig. 4). The mean change in consistency score of the glands estimated by rectal palpation remained unremarkable from the first to the last examination (P = 0.63); however, in three bulls the consistency score decreased from 4 to 1 by the time of the last examination. A high correlation was found between semen density and gross motility (r = 0.51; P < 0.0001), gross and individual motility (r = 0.68; P < 0.001), percentage of live sperm and individual motility (r = 0.64; P < 0.0001), and between individual motility and percentage of normal sperm (r = 0.46;
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Fig. 4. Mean scores representing the consistency of the vesicular glands and the presence of pus, leukocytes and blood in the semen samples obtained before and after intraglandular antibiotic injection from all bulls affected with vesicular adenitis. Semen samples were collected every 4 days. Letters a and b means of scores with different superscripts differ (P < 0.05).
P < 0.0001). There was a significant negative correlation between the presence of leukocytes in semen and percentage of live sperm (r = −0.24; P < 0.02), and between leukocytes and the percentage of midpiece defects (r = −0.24; P = 0.02). There was no difference from the first to last semen samples, before and after treatments, in sperm morphology (P = 0.24); however, motility improved when pus was no longer present in semen (gross motility, P = 0.05; individual motility, P < 0.08). When inflamed and healthy vesicular glands were compared by ultrasonography before and 1 wk after treatment, there was no effect of gland health status, time or health status by time interaction on the MN (P = 0.3, P = 0.46, and P = 0.64, respectively) or S.D. (P = 0.16, P = 0.77, and P = 0.20, respectively) of pixel distribution. 4. Discussion This is the first report of studies on intraglandular injections of antibiotics in beef bulls using the ischiorectal fossa as a route of administration and the first report of ultrasonographic pixel characterization of inflamed and healthy vesicular glands with or without intraglandular antibiotic treatment. Previous studies have focused mainly on the description of pathogenesis, pathological findings and diagnosis rather than treatment of vesicular adenitis (Dargatz et al., 1987; Galloway, 1964; Blom, 1979a,b). The pararectal approach for gland injection allowed good control of the positioning of the needle for gland injection and no appreciable adverse reaction was found after this procedure in the animals’ behavior or by rectal palpation and ultrasonography. In the present study, the
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type of intraglandularly injected antibiotic influenced the volumetric size of vesicular glands. A greater glandular volume was observed in those treated with oxytetracycline chlorhydrate or penicillin than in those not treated or treated with ceftiofur. It appeared that ceftiofur induced less inflammation and might be the more suitable antibiotic for intraglandular treatment. Adverse consequences of greater inflammation could include the development of adhesions between the gland and surrounding tissue as has been observed previously when iodine solution was used to treat the condition (Hoover, 1974). The lesser degree of inflammation provoked by ceftiofur is most likely beneficial clinically and on welfare grounds. The proposed differences in tissue reaction induced by the different antibiotics were also supported by the differences in pixel MN values that were observed over time. Ultrasonography has been used for pixel analysis of testicular tissue in bulls (Aravindakshan et al., 2000; Brito et al., 2003) and testes and accessory glands of growing healthy bull calves (Chandolia et al., 1997), but the use of ultrasonography to study the effect of an antibiotic injection into the vesicular glands has not been previously reported. Simple visual observations of ultrasonograms revealed a dramatic difference among treatments in gland size and echogenicity. Computer analysis of pixel intensity distribution in ultrasonograms confirmed the visual observations of increased echogenicity of vesicular glands after injection with antibiotics. A remarkable change was noticed in the mean pixel intensity of ultrasonograms taken immediately after antibiotic injection. The mean pixel intensity value MN was greater in glands treated with oxytetracycline and penicillin compared to untreated glands or those treated with ceftiofur. Each antibiotic solution might have had differences in echogenicity from other antibiotics and this characteristic might have affected the tissue echogenicity immediately after injection. However, a direct measurement of the pixel intensities of antibiotic solutions was not conducted. Oxytetracycline- and penicillin-treated glands continued to have a higher percentage of MN compared to ceftiofur in ultrasonographic scans at 24, 72 and 168 h after treatment. This continued increase in pixel intensity concurs with findings on rectal palpation of glands in the post-treatment period when antibiotic-treated glands felt enlarged and firmer in texture. The pixel MN returned to pre-treatment level values 7 days after treatment with ceftiofur, whereas, the pixel MN for oxytetracline- and penicillin-treated glands remained elevated. This would indicate that ceftiofur was less irritating to the tissue than the other antibiotics. In bulls with vesiculitis, there was no change in the pixel intensity in glands from pretreatment scans to ultrasound examinations 7 days after treatment. Injection of antibiotic into the glands resulted in the presence of blood in semen samples regardless of the type of antibiotic used. Presumably bleeding occurred due to tearing of tissue at the site of antibiotic deposition. The presence of blood in semen samples declined to undetectable levels after 28 days and while tissue hemorrhage is undesirable in the short term, there appeared to be no lasting adverse effect. Intraglandular treatment did not adversely affect semen quality as sperm morphology did not change over the period of treatment and there was a beneficial effect on sperm viability after pus was no longer present in semen. The antibiotic distribution throughout the vesicular gland after direct injection might be a concern. Even though glands were always injected approximately in the middle, immediate ultrasound scans showed that the antibiotic did not reach some peripheral areas of the glands. This could only be assessed in the oxytetracycline- and penicillin-treated groups because these antibiotics caused greater gland tissue echogenicity than ceftiofur. In addition, if the tip of the needle was placed in or near a large glandular duct there could be loss of antibiotic from the injection site through a duct. Another important aspect in the glandular distribution of the antibiotic is the nature of the antibiotic. The third generation of cephalosporins, to which ceftiofur belongs, and procaine peni-
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cillin G have an extensive distribution in extracellular fluid, but the passage across membranes or other physiological barriers is poor (Prescott, 2002a,b). Penetration of physiological barriers by penicillin is increased in inflammatory processes (Prescott, 2002b). This may have been the cause of success in the intraglandular treatment of infected glands in a bull in Experiment 2. A. pyogenes was the most common kind of bacterium isolated from the infected bulls and in three cases was associated with a concomitant bacterium. It has also been the most common isolate reported in the literature (Cavalieri and Van Camp, 1997; Carroll et al., 1968; Phillips, 1993; Mickelsen et al., 1994; Carson, 2000). Blom and Christensen (1965) reported that A. pyogenes was cultured in 27.4% of 6211 semen samples examined while Escherichia coli was isolated from 20% of semen samples. In the present study, E. coli was isolated in three bulls. A particularly interesting finding was the isolation from one bull of Corynebacterium pseudotuberculosis which has not been reported previously in cases of vesicular adenitis. This bull received only one treatment with ceftiofur and in the subsequent semen collections showed a significant improvement. There were also other Corynebacteria isolated from semen samples. In most cases, Corynebacterium sp. were cultured in association with other bacteria such as Pasteurella sp. and A. pyogenes. A. pyogenes was found in 37.7% of 106 preputial washings while Corynebacterium renale and other Corynebacterium sp. were found in 53.8% (Galloway, 1964). These bacteria were also cultured from vesicular glands obtained post-mortem. This led to speculation that bacteria that usually colonize the prepuce and penis may cause vesicular adenitis using the hematogenous route of infection through small lacerations in the glans penis when bulls mount each other. Isolation of Streptococcus sp. in a small percentage of bulls with vesiculitis has been reported previously (Ball et al., 1968), and was seen in one of the 14 bulls in the present study. In other published cases of vesicular adenitis, at times no bacteria could be isolated. In the present study, no bacteria were cultured from three bulls from the same farm. This might have been due to unreported antibiotic treatment of the bulls before they were submitted for the present experiment. Due to a limited availability of clinical cases and limited funding for the transport and care of bulls, an untreated control group was not used. This is a shortcoming of Experiment 2 because an untreated control group was necessary to verify that the treatments used were more beneficial than no treatment. Further studies are needed to verify the conclusions of the present study. 5. Conclusion The present experiments indicated that intraglandular injection of ceftiofur or penicillin in beef bulls via the ischiorectal fossa appeared to be safe and effective in the treatment of vesicular adenitis. Ultrasonography can be used to evaluate the characteristics of the glands prior to and after intraglandular antibiotic treatment. Evaluation of vesicular glands by rectal palpation is also useful in monitoring the effect of treatments, but semen collection is likely the most useful method to evaluate the progress of recovery from vesiculitis. The present study also gave information about the etiology of vesiculitis of western Canadian bulls and it constitutes the first report of isolation of C. pseudotuberculosis from a case of vesicular adenitis in a bull. References Al-Aubaidi, J.M., McEntee, K., Lein, D.H., Roberts, S.J., 1972. Bovine seminal vesiculitis and epididymitis caused by Mycoplasma bovigenitalium. Cornell Vet. 59, 589–596. Aravindakshan, J.P., Honaramooz, A., Bartlewski, P.M., Beard, A.P., Pierson, R.R., Rawlings, N.C., 2000. Gonadotrophin secretion in prepubertal bull calves born in spring and autumn. J. Reprod. Fertil. 120, 159–167.
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