Campylobacter jejuni is a small (width, 0.2 to 0.9 μm; length, 0.2 to 5.0 μm), spiral, gramnegative, non–spore-forming bacterium that is vigorously motile owing to a polar flagellum at 1 or both ends.1 Campylobacter species are a leading cause of reproductive losses in sheep, with abortion rates that range from 5% to 50% in infected flocks.2–4 Historically, Campylobacter-induced abortions in sheep were primarily associated with Campylobacter fetus ssp fetus and to a lesser extent with Campylobacter jejuni.1,3 However, over the past few decades, C jejuni has replaced C fetus ssp fetus as the predominant cause of abortion in sheep in the United States. This etiologic species shift appears to be primarily driven by the expansion of a hypervirulent, tetracycline-resistant C jejuni clone, termed the SA clone. The SA clone is currently responsible for 93% of C jejuni abortions in sheep in the midwestern United States.1,2
During outbreaks of Campylobacter-induced abortions in sheep, antimicrobials may be administered in the feed to facilitate treating large numbers of animals over an extended period of time. In the United States, the only feed-grade antimicrobial currently approved by the FDA for the prevention or treatment of Campylobacter-induced abortion in sheep is chlortetracycline at a rate of 80 mg/sheep/day.5 Pharmacokinetic studies5,6 indicate that feeding chlortetracycline to sheep at the FDA-approved dose, or at an even higher dose (500 mg/sheep/day), results in drug concentrations that are presumed to be subtherapeutic in the plasma of pregnant ewes and are generally undetectable in fetal tissues and amniotic fluid. Moreover, most recent C jejuni isolates obtained from US sheep are resistant to tetracycline, largely owing to the presence of the tet(O) gene,2,7,8 Thus, for sheep flocks, administration of chlortetracycline via the feed is unlikely to be effective during abortion outbreaks caused by C jejuni.
Results of susceptibility testing for C jejuni isolates cultured from field cases of SAs indicate that most are susceptible to macrolides, including tulathromycin, azithromycin, telithromycin, and erythromycin.2,7,8 Tulathromycin is a recently developed, semi-synthetic, triamilide macrolide.9 The benefits of treating sheep with tulathromycin include its ease of administration (SC injection), wide volume of distribution, low therapeutic concentrations, and long terminal half-life, which ranges from 60 to 140 hours across domestic species.9 The mean ± SD apparent elimination half-life of tulathromycin is 110.8 ± 20.9 hours in pregnant ewes.10 The protracted duration of action for tulathromycin following a single injection to pregnant ewes might be protective during an abortion outbreak caused by C jejuni. Additionally, tulathromycin administration to pregnant ewes results in detectable concentrations of the drug in fetal plasma and amniotic fluid that persist for days.10
To our knowledge, the only experimental model for C jejuni-induced abortion in sheep involves IV inoculation of the bacterium.11,a Most pregnant ewes will abort 3 to 12 days after IV administration of C jejuni.11,a Characteristics of experimental C jejuni-induced abortions in sheep include suppurative metritis, placentitis with numerous intralesional bacterial colonies, and the isolation of large numbers of the bacterium from uterine, placental, and fetal tissues.11,a However, that experimental model represents a particularly aggressive C jejuni challenge to pregnant ewes and is associated with some adverse effects. In a small proportion of ewes, IV inoculation of C jejuni can cause severe endotoxemia, which may necessitate euthanasia within 24 hours after injection.a Other ewes may develop vaginal bleeding or abort within 24 to 48 hours after C jejuni inoculation owing to the effects of the bacterial endotoxin on the fetus and placenta.a The purpose of the study reported here was to evaluate the efficacy of tulathromycin for the prevention of abortion in pregnant ewes when administered within 24 hours after experimental inoculation with C jejuni.
Materials and Methods
Animals
All study procedures were reviewed and approved by the Iowa State University Institutional Animal Care and Use Committee. Twenty, primiparous Hampshire-Polypay crossbred ewes were acquired from the Iowa State University Sheep Teaching Facility. The flock maintained at the teaching facility had not had any infectious abortions reported for several years prior to study initiation. The study ewes underwent an estrus synchronization protocol and were bred by natural service in accordance with the teaching flock's standard operating procedure. Transabdominal ultrasonography was used to confirm pregnancy and estimate the stage of gestation for all study ewes. All ewes were estimated to be between 72 and 92 days of gestation at the time of acquisition for the study. The ewes were transported to the Iowa State University Laboratory Animal Resources research facility where they were individually weighed and had ear tags applied for identification purposes. The ewes were housed in pairs in pens with plastisol-coated steel mesh flooringb in a biosafety level-2 facility, and had ad libitum access to water and a complete ration designed for small ruminants.c Ewes were allowed to acclimate to the facility for 3 days before C jejuni inoculation.
C jejuni inoculation
Campylobacter jejuni IA3902, a clinical strain of an SA clone originally isolated from an aborted ovine fetus, was used as the inoculant for all sheep. The isolate was confirmed to be an SA clone on the basis of results of pulsed-field gel electrophoresis, multi-locus sequence typing, cmp gene sequence typing, and whole genome sequencing as described.2,12 Fresh cultures of the bacterium were created to inoculate the sheep. Briefly, the isolate was inoculated onto MH agar in anaerobic jars and incubated for 24 hours under microaerobic conditions (ie, 5% oxygen, 10% carbon dioxide, and 85% nitrogen) at 42°C. Campylobacter jejuni was harvested from the MH agar and washed once with PBS solution to remove free endotoxin. The harvested bacteria were then suspended in sufficient sterile PBS solution to achieve the desired concentration (8.5 × 108 CFUs/mL), which was determined on the basis of the optical density of the suspension measured at a wavelength of 600 nm. The final number of C jejuni organisms in the suspension was determined by counting the number of viable CFUs.
All 20 study ewes were inoculated with C jejuni. The inoculation was performed with each ewe unsedated and manually restrained. The cranial half of the left jugular furrow was shaved to remove the wool. The underlying skin was aseptically prepared in a routine manner by the use of 3 alternate applications of 2% chlorhexidine gluconate scrub and 70% isopropyl alcohol. An 18-gauge, 2-inch catheter was placed in the left jugular vein. Because body weight varied among the ewes (body weight range, 46 to 75.5 kg), graded doses of the C jejuni inoculant were administered. Ewes that weighed between 45 and 54.5 kg were administered 1 mL of the inoculant (8.5 × 108 CFUs of C jejuni), ewes that weighed > 54.5 to 64 kg were administered 1.15 mL of the inoculant (9.8 × 108 CFUs of C jejuni), and ewes that weighed > 64 kg were administered 1.25 mL of the inoculant (10.6 × 108 CFUs of C jejuni). The designated amount of the C jejuni suspension was injected into the catheter with a syringe. After the injection was complete, a small amount of blood was aspirated back into the syringe and reinfused to ensure that the entire inoculum was administered.
Treatment
Ewes were first stratified into 2 groups on the basis of the number of days in gestation (≥ or < 70 days). Ewes were further subdivided on the basis of median body weight (≥ or < 60.5 kg). Then, ewes from these four stratified groups were randomly assigned by means of a random number generator to receive either tulathromycind (1.1 mL/45 kg [2.4 mg/kg]) or an equal volume of sterile isotonic saline (0.9% NaCl) solution (1.1 mL/45 kg; sham treatment) such that there were 10 sheep in each treatment group. The designated treatment was injected SC over the thorax in the region anterior to the axilla 18 hours after C jejuni inoculation.
Ewe monitoring
All ewes were monitored twice daily for signs of depression, hyporexia, prolonged recumbency or reluctance to rise, fever, and evidence of impending (vaginal bleeding) or actual abortion. Any ewe that exhibited signs of marked depression or became recumbent (unable to rise) following C jejuni inoculation was immediately euthanized for humane reasons. Ewes that aborted or had signs of vaginal bleeding were also immediately euthanized. The remaining ewes were euthanized 21 days after C jejuni inoculation. All ewes were euthanized with a penetrating captive bolt device in accordance with the AVMA Guidelines for the Euthanasia of Animals.13 Briefly, for each ewe following application of the captive bolt device and loss of consciousness and the corneal reflex, pneumothorax was created as an adjunct method to ensure death. Each ewe was necropsied immediately after death was confirmed.
Necropsy
Each ewe was inspected for gross lesions and specimens were collected for bacterial culture and histologic examination during necropsy. Specimens submitted for bacterial culture included a swab of the cecal contents, heart blood, bile, uterus, placentomes (ewes that did not abort) or placenta (ewes that aborted), and a homogenate of fetal lung and liver tissues. Bile samples from each ewe were collected directly from the gallbladder with a sterile tuberculin syringe and 26-gauge, 3/8-inch needle. Specimens of the uterus, placentome or placenta, and fetal lung-liver tissue homogenate were placed in separate sterile Petri dishes. All samples and specimens designated for bacterial culture were refrigerated immediately after collection, and cultures were set up the same day. Specimens collected for histologic examination included liver, gallbladder, placentome (ewes that did not abort) or placenta (ewes that aborted), and fetal lung and liver. All specimens designated for histologic examination were fixed in neutral-buffered 10% formalin for 24 hours and then transferred to a 70% ethanol solution. Specimens were then trimmed and processed in a routine manner and stained with H&E stain for histologic evaluation. Additionally, sections of placentome and placenta were stained with Gimenez stain for assessment of intracytoplasmic organisms consistent with Coxiella burnetii or Chlamydophila spp.
Culture for Campylobacter spp
For culture of Campylobacter spp and semiquantitative assessment of C jejuni, samples were streaked onto MH agar that contained Preston Campylobacter-selective supplemente (trimethoprim, rifampicin, polymyxin B, and cycloheximide) and Campylobacter growth supplemente (sodium metabisulfite, sodium pyruvate, and ferrous sulfate). Cecal swab specimens were streaked directly onto an agar plate. A sterile cotton swab was used to streak a small amount (approx 0.1 mL) of bile or blood onto an agar plate. Tissue specimens (uterus, placentome, placenta, and pooled fetal lung and liver tissues) were minced with sterile scissors or scalpels and then streaked onto an agar plate with a sterile inoculation loop. Inoculated agar plates were incubated for 48 hours in anaerobic jars under microaerobic conditions at 42°C. For each plate following incubation, Campylobacter-like colonies were counted to estimate the number of CFUs in each sample. One suspect colony from each sample or animal underwent further species identification by means of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry as described.14,15
Histologic evaluation
For each ewe, 2 sections of uterine tissue were histologically evaluated and abnormalities were scored on a 4-point scale, where 0 = no inflammatory infiltrate, 1 = mild suppurative inflammation in the superficial submucosa and mild suppurative exudate within scattered uterine glands, 2 = moderate suppurative inflammation in the superficial submucosa and moderate suppurative exudate within scattered uterine glands, and 3 = marked suppurative inflammation in the superficial submucosa and marked suppurative exudate within scattered uterine glands. Additionally, 4 sections of cotyledons or placentomes were evaluated and abnormalities were scored on a 5-point scale, where 0 = no identifiable inflammation or bacterial colonies, 1 = mild focal to multifocal suppurative inflammation involving < 10% of the cotyledon or placentome with absent to rare bacterial colonies, 2 = moderate multifocal suppurative inflammation involving 10% to 50% of the cotyledon or placentome with occasional bacterial colonies, 3 = moderate to severe multifocal suppurative inflammation involving 50% to 75% of the cotyledon or placentome with moderate numbers of bacterial colonies, and 4 = suppurative inflammation involving > 75% of the cotyledon or placentome with numerous bacterial colonies.
Data analysis
Each ewe was classified as having 1 of 3 outcomes. The nonabortion category included ewes that did not abort or for which vaginal bleeding indicative of impending abortion was not observed during the 21-day observation period. The endotoxin-related termination category included ewes that became systemically ill within 12 hours and were euthanized or aborted within 48 hours after C jejuni inoculation. Ewes in this category had gross evidence of endothelial damage characterized by hemorrhages or effusions but no histologic evidence of placentitis or endometritis, and culture of uterine, placental, and fetal tissues yielded no growth of Campylobacter spp. The Campylobacter abortion category included ewes that aborted or developed vaginal bleeding indicative of impending abortion and were euthanized before the end of the 21-day observation period. Ewes in this category had histologic evidence of placentitis and endometritis with no C burnetii or Chlamydophila spp observed on Gimenez-stained tissue specimens in addition to heavy growth of Campylobacter spp on cultures of uterine, placental, and fetal tissue specimens.
Descriptive statistics were generated for each outcome category. The proportion of ewes that developed vaginal bleeding or aborted and the proportion of ewes with Campylobacter spp isolated from culture of cecal contents were compared between the 2 treatment groups by means of χ2 tests.
Results
One sham-treated ewe and 1 tulathromycin-treated ewe developed signs of depression and became tachypneic and recumbent within 24 hours after C jejuni inoculation. Both ewes were euthanized and categorized in the endotoxin-related termination outcome category. During necropsy, both ewes had blood-colored froth draining from the nostrils, moderate serosanguinous thoracic effusion, and moderate multifocal pulmonary hemorrhage. The fetuses of one ewe had multifocal pulmonary hemorrhage, and the fetus of the other ewe had focally extensive hepatic hemorrhage. Histologic evidence of inflammation was not present in the uterus or placentomes of either ewe, and no Campylobacter spp were isolated from the uterus, placenta, or fetal tissues. Those 2 ewes were excluded from all χ2 tests.
Seven sham-treated ewes and 2 tulathromycin-treated ewes were categorized in the Campylobacter abortion outcome category. The 7 sham-treated ewes developed vaginal bleeding or aborted and were euthanized between 4 and 21 days after C jejuni inoculation. One of the 2 tulathromycin-treated ewes developed vaginal bleeding, the other aborted, and both were euthanized 6 days after C jejuni inoculation. The tissues from all 9 ewes in this outcome category had histologic evidence of severe metritis (grade, 3) and placentitis (grade, 4), and numerous Campylobacter-type bacteria were observed in the placental tissue. Gimenez-stained placental tissue sections did not have any evidence of microorganisms consistent with C burnetii or Chlamydophila spp. Large numbers of C jejuni were isolated from the uterine, placental, and fetal tissues of all 9 ewes. Campylobacter organisms were not isolated from the blood of any ewe but were isolated from the bile of 1 sham-treated ewe and 1 tulathromycin-treated ewe, both of which aborted.
Two sham-treated and 7 tulathromycin-treated ewes did not develop vaginal bleeding or abort during the 21 days after C jejuni inoculation and were categorized in the nonabortion outcome category. One tulathromycin-treated ewe had histologic evidence of mild (grade, 1) multifocal inflammation in the placentome and mild (grade, 1) metritis, and low numbers of C jejuni were cultured from uterine, placental, and fetal tissues. Tissues from both sham-treated ewes and the remaining 6 tulathromycin-treated ewes lacked histologic evidence of metritis or placentomitis. Campylobacter organisms were not cultured from the uterus, placentome, or fetal tissues of any of those 8 ewes.
The proportion of tulathromycin-treated ewes that developed vaginal bleeding or aborted during the 21 days after C jejuni inoculation (2/9) was significantly less than that for the sham-treated ewes (7/9). Similarly, the proportion of tulathromycin-treated ewes from which Campylobacter organisms were cultured from cecal swab specimens obtained during necropsy (1/9) was significantly less than that for the sham-treated ewes (7/9).
Discussion
Numerous procedures are recommended for sheep flocks during an abortion outbreak caused by Campylobacter spp including prompt disposal of aborted fetuses and placenta, separation of pregnant ewes from ewes that have aborted, removal of pregnant ewes from the contaminated environment, and the implementation of appropriate hygienic protocols to prevent spread of the organism via contaminated clothing, boots, and equipment.16 Antimicrobial administration may also be considered to help minimize ongoing losses in potentially exposed ewes.16 In recent years, most C jejuni isolates cultured from field cases of SAs have been susceptible to macrolides, including tulathromycin.2,7,8 In the present study, the proportion of pregnant ewes that developed vaginal bleeding or aborted after being experimentally inoculated with C jejuni was significantly less for ewes that received tulathromycin (2.4 mg/kg, SC), compared with ewes that received a sham treatment (sterile saline solution) within 24 hours after C jejuni inoculation. That finding suggested that administration of tulathromycin to pregnant ewes in the midst of an abortion outbreak caused by C jejuni might help prevent further abortions. However, it is important to note that abortion was not prevented in all tulathromycin-treated ewes of the present study, likely owing to the aggressive nature of the experimental C jejuni inoculation (8.5 × 108 to 10.6 × 108 CFUs, IV).
Primary sources of Campylobacter organisms during an abortion outbreak include aborted fetuses and placenta, uterine discharge, and the contaminated environment. Bacterial culture results of the present study confirmed that uterine, placental, and fetal tissues were heavily colonized by C jejuni. Campylobacter organisms can colonize the intestinal tract and gallbladder of infected animals, and fecal shedding from infected animals can be an important source of the bacteria.1,7 In the present study, the proportion of tulathromycin-treated ewes from which Campylobacter organisms were cultured from cecal swab specimens (1/9) was significantly less than that for the sham-treated ewes (7/9). Thus, another potential benefit of tulathromycin administration to C jejuni– infected ewes is a decrease in the number of organisms shed in the feces.
In the present study, one of the tulathromycin-treated ewes that did not abort during the 21-day observation period following C jejuni inoculation had mild metritis and placentomitis, and small numbers of C jejuni were cultured from the uterus, placentome, and fetal tissues. Had that ewe not been euthanized at the end of the observation period, it might have eventually aborted or delivered stillborn or weak lambs. Further research is necessary to determine whether administration of a second dose of tulathromycin 10 to 14 days after the first would have eliminated such an infection.
Administration of tulathromycin to pregnant ewes to prevent abortion caused by C jejuni represents extralabel drug use and requires a valid veterinarian-client-patient relationship, appropriate veterinary oversight and documentation, and the observation of prolonged milk and meat withdrawal intervals. Veterinarians should evaluate available diagnostic information and consider principles of prudent antimicrobial use when considering whether tulathromycin administration is appropriate for prevention of C jejuni abortion in sheep. It is important to note that the present study was not designed to evaluate, nor was it intended to advocate, the use of tulathromycin as a prophylactic treatment for C jejuni–induced abortion in sheep. Prophylactic use of the drug may facilitate the development of tulathromycin-resistant bacteria.17 Currently, macrolides appear to be effective against most strains of C jejuni associated with abortion in sheep. However, macrolides are often the drug of choice for the treatment of campylobacteriosis in both human and veterinary medicine, and their widespread use has hastened the emergence of macrolide resistance amongst Campylobacter strains.18 Consequently, prophylactic use of tulathromycin in sheep is not recommended.
Acknowledgments
Supported by USDA APHIS Cooperative Agreement AP17VSSPRS00G002. The funding source did not have any involvement in the study design, data analysis and interpretation, or writing and publication of the manuscript.
Dr. Ocal was supported by the International Postdoctoral Research Scholarship Program 2219 (No. 1059B191700841) of the Scientific and Technological Research Council of Turkey.
ABBREVIATIONS
| MH | Mueller-Hinton |
| SA | Sheep abortion |
Footnotes
Lashley V, Yaeger M, Sahin O, Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, Iowa: Unpublished data, 2019.
Tenderfoot flooring system, Tandem Products Inc, Minneapolis, Minn.
Teklad 7060, Envigo, Huntingdon, England.
Draxxin (tulathromycin concentration, 100 mg/mL), Zoetis Inc, Kalamazoo, Mich.
Oxoid Microbiology Products, Thermo Fisher Scientific, Waltham, Mass.
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