Pathology in Practice

Jessica I. Hanlon 1Animal Disease Diagnostic Laboratory, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.
2Department of Comparative Pathology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.

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José A. Ramos-Vara 1Animal Disease Diagnostic Laboratory, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.
2Department of Comparative Pathology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.

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Samuel L. Yingst 1Animal Disease Diagnostic Laboratory, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.
2Department of Comparative Pathology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.

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G. Kenitra Hendrix 1Animal Disease Diagnostic Laboratory, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.
2Department of Comparative Pathology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.

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History

Two aborted fetuses (A and B) from a 4-year-old ewe were submitted for an abortion evaluation. Fetuses A and B had a crown-to-rump length of 41.8 cm and 42.5 cm, respectively, indicating a gestational age each of approximately 19 to 21 weeks. This ewe was from a group of approximately 40 ewes. In this flock, 3 other ewes aborted; 1 ewe aborted midgestation and the other 2 ewes aborted around the same date and stage of gestation as this ewe. Another ewe in this flock gave birth to twins, one of which was stillborn and one that was weak but survived. An additional ewe gave birth to a pair of weak lambs that died on the day of parturition. None of the ewes had any clinical signs. The first abortion occurred midgestation; the remainder of the abortions, the still-birth, and the birth of the weak lambs all occurred within a 3-week period. The ewes in this flock were typically bred naturally or via embryo transfer. No new sheep had entered this flock recently, and the ram used for breeding was from the same farm. This farm had not had any major ovine reproductive tract issues in the past.

Gross Findings

Approximately 50% of the fetal membranes from fetus A had multifocal to coalescing, variably sized, irregular, pale tan, firm, raised plaques adhered to the cotyledons, intercotyledonary areas, and the umbilicus. The rims of other cotyledons in the less affected areas were pale tan and raised; in those areas, the intercotyledonary areas had a smooth and glistening surface (Figure 1). There were multifocal, 1- to 3-mm-diameter, raised, dark red to black, lightly adhered plaques on the intercotyledonary areas of the fetal membranes from fetus B. No gross lesions were evident in the remainder of the tissues of fetus A or B.

Figure 1—
Figure 1—

Photographs of the fetal membranes associated with 1 of 2 fetuses (fetus A) aborted at a gestational age of approximately 19 to 21 weeks by a ewe with no signs of clinical disease. A—Notice the multifocal to coalescing, pale tan plaques of fibrinonecrotic material that coats the cotyledons and intercotyledonary areas (most noticeably in the left half of the placenta). The intercotyledonary areas on the right side are not affected (circles). B—In a higher-magnification view, the inflammatory exudate is visible on the rim of the 2 cotyledons (asterisks) and on the intercotyledonary areas (circles). In both panels, bar = 5 cm.

Citation: Journal of the American Veterinary Medical Association 255, 8; 10.2460/javma.255.8.903

Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page→

Histopathologic and Molecular Findings

Microscopic examination revealed that the chorionic villi of the fetal membranes from fetus A were multifocally necrotic and had areas of mineralization. Trophoblasts were expanded by numerous, intracytoplasmic, basophilic coccobacilli (Figure 2). Many degenerate neutrophils, fewer macrophages, and fibrin had infiltrated into the stroma of both the cotyledonary and intercotyledonary areas. The intercotyledonary stroma was expanded by edema. Vessel walls were infiltrated and surrounded by macrophages, degenerated neutrophils, lymphocytes, plasma cells, and fibrin (vasculitis). The umbilical surface was multifocally infiltrated by many degenerated neutrophils and fewer macrophages. Giemsa staining was performed on a section of the fetal membranes from fetus A; the bacteria expanding the trophoblasts were Giemsa stain positive. Similar lesions were observed in sections of the fetal membranes from fetus B. No microscopic lesions in other fetal tissues were identified.

Figure 2—
Figure 2—

Photomicrographs of sections of the fetal membranes from fetus A. A—Section of a cotyledon. Villi (v) are partially lined by trophoblasts colonized by numerous, intracytoplasmic, basophilic, coccobacilli (arrows). The chorion (circles) is expanded by edema and mixed infiltrates of leukocytes. There is fibrinosuppurative exudate (asterisk) with dystrophic mineralization on the surface of the cotyledon. H&E stain; bar = 1,250 μm. B—Higher-magnification view of the affected chorionic villi. Many trophoblasts (arrows) are markedly enlarged by basophilic, intracytoplasmic bacteria. Some trophoblasts and chorionic villi are necrotic (n). Infammation (I) is also observed. H&E stain; bar = 120 μm. Inset—High-magnification view of trophoblasts infected with intracytoplasmic bacteria. H&E stain; bar = 25 μm.

Citation: Journal of the American Veterinary Medical Association 255, 8; 10.2460/javma.255.8.903

An abortion panel (that included bacteriologic, virologic, and molecular diagnostic testing) was performed on samples from each of the submitted fetuses and the fetal membranes. Results of a Coxiella burnetii-specific PCR assay (targeting the IS1111 gene1) were positive. Bacterial cultures of fetal membrane samples yielded no notable growth; results of viral testing (for bluetongue virus and pestivirus) and a molecular panel (real-time PCR assays specific for Chlamydia abortus, Chlamydia psittaci, Brucella spp, Leptospira spp, Toxoplasma gondii, and Neospora caninum) were also negative. Twenty-five ewes had given birth within a 30-day period after submission of fetuses A and B for investigation. A vaginal swab specimen collected from each of these ewes underwent C burnetii-specific PCR assay; results were positive for C burnetii for 17 of the 25 ewes.

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: fibrinosuppurative infection of fetal membranes with intratrophoblastic bacteria (fetuses A and B) and suppurative funisitis (fetus A).

Case summary: Coxiella-associated placentitis in a ewe that resulted in abortion of 2 fetuses.

Comments

Coxiella burnetii is a gram-negative, zoonotic, obligate intracellular bacterium found in many countries.2–7 Coxiella burnetii was discovered in 1937 as a cause of fever in 20 of 800 meat factory workers in Australia.2 The disease was termed query fever at the time because of its unknown etiopathogenesis. Rickettsia burnetii (now known as C burnetii) was isolated from samples of the affected Australians’ blood and urine.2,8 This disease is now known as Q fever in humans and as coxiellosis in other animals.9

The primary reservoirs of C burnetii include goats, cattle, and sheep; however, the organisms can also infect rodents, birds, arthropods (mainly ticks), cats, dogs, rabbits, wildlife species, and humans.2,8,9 This organism is present in the placenta and vaginal fluids; it is also spread by infected animals through urine, feces, milk, semen, and sputum.9,10 The most common routes of infection are inhalation of dust that has been contaminated with C burnetii from one of the aforementioned tissues or fluids and oral transmission when there is heavy contamination of the environment.3,7

Coxiella burnetii infection in ruminants may be subclinical; however, clinical signs can include abortion (typically during the end of the gestation period), stillbirths, and birth of weak progeny.2,4,7 The fetal membranes of an aborted fetus are typically thick and leathery and have a pale tan to yellow exudate in the intercotyledonary areas that can spread to the cotyledons.3,7 When the cotyledons are affected, the first change is the development of a rim of pale tan to white exudate with multifocal areas of similar exudate in the center.7 These fetal membrane lesions may be seen in animals that aborted and those that had a normal parturition because infected pregnant animals that actively shed this organism do not always have abortions.3,10 The aborted fetuses themselves are usually unremarkable; however, they may be autolyzed.5

When C burnetii infects a pregnant small ruminant, the primary target is the allantochorionic trophoblasts.3 Histologically, the trophoblasts of both the intercotyledonary areas and cotyledonary villi may be expanded by numerous, basophilic organisms.5,8 The chorioallantois tends to be inflamed; this inflammation ranges from a mild mononuclear placentitis to severe suppurative placentitis with necrosis.5,8 Placental vasculitis (as identified from the fetal membranes of fetuses A and B described in the present report) is not typical of C burnetii infection, but when present, does not exclude C burnetii infection as a differential diagnosis.7 Leukocytoclastic vasculitis, which develops when immune complexes are deposited in vessel walls leading to neutrophil infiltration, has been reported in humans infected with C burnetii11; this may have been the cause of the placental vasculitis in the dam of fetuses A and B. If fetal lesions are present, they typically include mild changes such as granulomatous hepatitis and nonsuppurative pneumonia.7,8

Several diagnostic tests to detect C burnetii are available. An aborted fetus can be submitted (preferably with the fetal membranes) to a diagnostic laboratory and undergo an abortion panel. If fetal membranes are submitted, a PCR assay can be performed on tissue samples to test for C burnetii DNA. In addition, the fetal membranes can be examined and evaluated for gross and histologic lesions. It should be noted that positive PCR assay results for fetal membrane samples do not necessarily indicate that C burnetii infection was the cause of the abortion; nevertheless, PCR assay has high sensitivity and specificity for detection of C burnetii infection.1,12,13 A positive PCR assay result must be interpreted along with the gross findings, histologic findings, and ancillary test results.12 Also, because C burnetii is widespread in the environment, it can be an environmental contaminant.8 Therefore, all clinical, gross, histologic, and molecular findings need to be considered prior to making the final diagnosis of abortion caused by C burnetii infection. Additionally, immunohistochemical analysis can be performed on fetal membrane samples to detect C burnetii antigen.13 Serologic evaluation (by means of an ELISA, indirect fluorescent antibody testing, or complement fixation) of paired serum samples for C burnetii-specific antibodies is another testing option. However, findings are not always reliable9,10,13; for example, a positive ELISA result does not confirm that an animal is actively shedding the bacteria, and an animal that is actively shedding may be seronegative at the time of testing.9,10 Finally, bacterial culture of appropriate specimens can be performed, but it requires specific isolation techniques (use of special media, cell cultures, or embryonated eggs) at a biosafety level 3 laboratory because of the high infectivity of C burnetii and its ability to persist in the environment.1,2,5

Parenteral treatment of herd animals with oxytetracycline during a period of abortions can suppress continued abortions; however, such treatment does not prevent the animals from becoming infected.10,13 Treatment is not recommended when abortions are not occurring.13 Vaccines against C burnetii are not yet available in the United States. Culling of affected animals from a herd is not usually recommended because of persistent environmental contamination (possibly for a period of years).9 When a positive test result for C burnetii is obtained for a herd in the United States, the state board of animal health is notified and it is recommended that the farm personnel follow procedures outlined in the guidance document created by the National Association of State Public Health Veterinarians and the National Assembly of State Animal Health Officials.9,13

The largest human outbreak of C burnetii infections occurred in the Netherlands from 2007 to 2010, during which time approximately 4,000 humans were infected as a result of infection in approximately 28 goat herds and 2 sheep flocks.6,13 People are most likely to become infected by inhalation of the organisms; however, they can also be infected through contact with the parturition fluids (risk of infection being higher for farmers and veterinarians who assist with parturition or sample handling) or ingestion of unpasteurized milk or dairy products.4,9 It is important to note that when airborne, these organisms can travel for more than a mile; therefore, humans can become infected without any direct exposure to C burnetii-positive animals.13 Once infected, the mean incubation period in humans is 20 days (range, 3 to 30 days).13 Approximately 50% of humans infected with C burnetii do not develop clinical signs9,13; clinically affected individuals may have a fever, chills, headache, weakness, vomiting, diarrhea, pneumonia, and other flu-like symptoms.9,13 Chronic infections develop in < 5% of the people infected; chronic infections may involve endocarditis, hepatitis, and vasculitis. People with a valvular prosthesis or cardiac abnormalities or who are immunosuppressed or have previously undergone valvular surgeries are more at risk for development of chronic infections.2,13 Treatment (preferably with doxycycline) is usually indicated for patients with symptoms of acute or chronic Q fever.2,13

References

  • 1. Panning M, Kilwinski J, Greiner-Fischer S, et al. High throughput detection of Coxiella burnetii by real-time PCR with internal control system and automated DNA preparation. BMC Microbiol 2008;8:77.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Gürtler L, Bauerfeind U, Blümel J, et al. Coxiella burnetii-pathogenic agent of Q (query) fever. Transfus Med Hemother 2014;41:6072.

  • 3. Roest H-J, van Gelderen B, Dinkla A, et al. Q fever in pregnant goats: pathogenesis and excretion of Coxiella burnetii. PLoS ONE. 2012;7:e48949.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Berri M, Souriau A, Crosby M, et al. Shedding of Coxiella burnetii in ewes in two pregnancies following an episode of Coxiella abortion in a sheep flock. Vet Microbiol 2002;85:5560.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Agerholm JS. Coxiella burnetii associated reproductive disorders in domestic animals—a critical review. Acta Vet Scand 2013;55:13.

  • 6. Roest HIJ, Ruuls RC, Tilburg JJHC, et al. Molecular epidemiology of Coxiella burnetii from ruminants in Q fever outbreak, the Netherlands. Emerg Infect Dis 2011;17:668675.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Schlafer DH, Foster RA. Abortion in sheep, goats, and cattle caused by Coxiella burnetii. In: Jubb, Kennedy and Palmer's pathology of domestic animals. Vol 3. St Louis: Elsevier, 2016;416417.

    • Search Google Scholar
    • Export Citation
  • 8. Van den Brom R, van Engelen E, Roest HIJ, et al. Coxiella burnetii infections in sheep or goats: an opinionated review. Vet Microbiol 2015;181:119129.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Anderson AD, Szymanski TJ, Emery MP, et al. Epizootiological investigation of a Q fever outbreak and implications for future control strategies. J Am Vet Med Assoc 2015;247:13791386.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Berri M, Souriau A, Crosby M, et al. Relationships between the shedding of Coxiella burnetii, clinical signs and serological responses of 34 sheep. Vet Rec 2001;148:502505.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Koh SS, Li A, Cassarino DS. Leukocytoclastic vasculitis presenting in association with Coxiella burnetii (Q fever): a case report. J Cutan Pathol 2018;45:7173.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Hazlett MJ, McDowall R, DeLay J, et al. A prospective study of sheep and goat abortion using real-time polymerase chain reaction and cut point estimation shows Coxiella burnetii and Chlamydophila abortus infection concurrently with other major pathogens. J Vet Diagn Invest 2013;25:359368.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Anderson A, Boyer T, Garvey A, et al. Prevention and control of Coxiella burnetii infection among humans and animals: guidance for a coordinated public health and animal health response. Arlington, Va: National Association of State Public Health Veterinarians and National Assembly of State Animal Health Officials, 2013;130.

    • Search Google Scholar
    • Export Citation
  • Figure 1—

    Photographs of the fetal membranes associated with 1 of 2 fetuses (fetus A) aborted at a gestational age of approximately 19 to 21 weeks by a ewe with no signs of clinical disease. A—Notice the multifocal to coalescing, pale tan plaques of fibrinonecrotic material that coats the cotyledons and intercotyledonary areas (most noticeably in the left half of the placenta). The intercotyledonary areas on the right side are not affected (circles). B—In a higher-magnification view, the inflammatory exudate is visible on the rim of the 2 cotyledons (asterisks) and on the intercotyledonary areas (circles). In both panels, bar = 5 cm.

  • Figure 2—

    Photomicrographs of sections of the fetal membranes from fetus A. A—Section of a cotyledon. Villi (v) are partially lined by trophoblasts colonized by numerous, intracytoplasmic, basophilic, coccobacilli (arrows). The chorion (circles) is expanded by edema and mixed infiltrates of leukocytes. There is fibrinosuppurative exudate (asterisk) with dystrophic mineralization on the surface of the cotyledon. H&E stain; bar = 1,250 μm. B—Higher-magnification view of the affected chorionic villi. Many trophoblasts (arrows) are markedly enlarged by basophilic, intracytoplasmic bacteria. Some trophoblasts and chorionic villi are necrotic (n). Infammation (I) is also observed. H&E stain; bar = 120 μm. Inset—High-magnification view of trophoblasts infected with intracytoplasmic bacteria. H&E stain; bar = 25 μm.

  • 1. Panning M, Kilwinski J, Greiner-Fischer S, et al. High throughput detection of Coxiella burnetii by real-time PCR with internal control system and automated DNA preparation. BMC Microbiol 2008;8:77.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Gürtler L, Bauerfeind U, Blümel J, et al. Coxiella burnetii-pathogenic agent of Q (query) fever. Transfus Med Hemother 2014;41:6072.

  • 3. Roest H-J, van Gelderen B, Dinkla A, et al. Q fever in pregnant goats: pathogenesis and excretion of Coxiella burnetii. PLoS ONE. 2012;7:e48949.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Berri M, Souriau A, Crosby M, et al. Shedding of Coxiella burnetii in ewes in two pregnancies following an episode of Coxiella abortion in a sheep flock. Vet Microbiol 2002;85:5560.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Agerholm JS. Coxiella burnetii associated reproductive disorders in domestic animals—a critical review. Acta Vet Scand 2013;55:13.

  • 6. Roest HIJ, Ruuls RC, Tilburg JJHC, et al. Molecular epidemiology of Coxiella burnetii from ruminants in Q fever outbreak, the Netherlands. Emerg Infect Dis 2011;17:668675.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Schlafer DH, Foster RA. Abortion in sheep, goats, and cattle caused by Coxiella burnetii. In: Jubb, Kennedy and Palmer's pathology of domestic animals. Vol 3. St Louis: Elsevier, 2016;416417.

    • Search Google Scholar
    • Export Citation
  • 8. Van den Brom R, van Engelen E, Roest HIJ, et al. Coxiella burnetii infections in sheep or goats: an opinionated review. Vet Microbiol 2015;181:119129.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Anderson AD, Szymanski TJ, Emery MP, et al. Epizootiological investigation of a Q fever outbreak and implications for future control strategies. J Am Vet Med Assoc 2015;247:13791386.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Berri M, Souriau A, Crosby M, et al. Relationships between the shedding of Coxiella burnetii, clinical signs and serological responses of 34 sheep. Vet Rec 2001;148:502505.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Koh SS, Li A, Cassarino DS. Leukocytoclastic vasculitis presenting in association with Coxiella burnetii (Q fever): a case report. J Cutan Pathol 2018;45:7173.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Hazlett MJ, McDowall R, DeLay J, et al. A prospective study of sheep and goat abortion using real-time polymerase chain reaction and cut point estimation shows Coxiella burnetii and Chlamydophila abortus infection concurrently with other major pathogens. J Vet Diagn Invest 2013;25:359368.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Anderson A, Boyer T, Garvey A, et al. Prevention and control of Coxiella burnetii infection among humans and animals: guidance for a coordinated public health and animal health response. Arlington, Va: National Association of State Public Health Veterinarians and National Assembly of State Animal Health Officials, 2013;130.

    • Search Google Scholar
    • Export Citation

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