Adult female goat.During 2004 and 2005, a goat farm in Northern Taiwan accounted episodes of severe abortion. Two flocks of goats had been introduced from central and Southern Taiwan about half a year prior to the incidence.
At necropsy, the lungs of fetus were mottled, patchy, dark purple to dark grayish red. The placenta was diffusely dark-red with the presence of some yellow to red turbid exudates on the surface of the cotyledonary and intercotyledonary areas. No other gross lesions were found.
Microscopically, the placenta had multifocal to locally extensive necrosis in the superficial epithelium in both cotyledons and intercotyledonary areas. The trophoblasts were distended by small, approximately 1μm diameter, basophilic, intracytoplasmic organisms. The underlying stroma had mild to moderate lymphoplasmacytic infiltrates with evident perivascular lymphoplasmacytic aggregates. Macchiavellos staining revealed heavy intracytoplasmic load of positive microorganisms that were negative for Gram stain. In the fetus, mild to moderate inflammation consisting of lymphocytes, plasma cells, and epithelioid macrophages are frequently seen within in the parenchyma of liver, lungs, and kidney. Occasionally, non-suppurative meningoencephalitis and lymphocytic perivascular cuffing could also be observed.
Placenta, chorioallantoic: Placentitis, necrotizing, acute, multifocal, moderate, with focal vasculitis and intracellular organisms.
1. The detection of Coxiella burnetii by PCR with the primer pair of Trans1/Trans2 (687 bp) (Houver et al., 1992) and by nested PCR with the primer pairs of OMP1/OMP2 (501 bp) and OMP3/OMP4 (438 bp) (Zhang GQ et al., 1998) was employed for the final diagnosis of this case.
2. Other laboratory tests including chlamydiosis by PCR, foot & mouth disease by ELISA, and bluetongue by ELISA were all negative. Bacterial culture of the fetal tissues was also negative.
Coxiella burnetii is a ubiquitous zoonotic pathogen of Q fever, initially identified in Queensland, Australia, in 1935, after an outbreak of febrile illness among slaughterhouse workers. Coxiella was historically considered as a Rickettsia, but gene-sequence analysis now classifies it in the order Legionellales, family Coxiellaceae, genus Coxiella. It is an intracellular, small pleomorphic gram-negative bacterium, which completes its life cycle within the phagosomes of infected cells. Although possessing a membrane similar to that of the gram-negative bacteria, it is usually not stained by the Gram technique. According to Raoult et al. (2005), the survival and multiplication of C. burnetii in the acidophilic phagosomes prevent antibiotics from killing the bacteria. Increasing pH with lysosomotropic agents such as chloroquine restores the bactericidal activity of doxycycline. The agent has 2 distinct life cycle stages known as the large-cell variant (LCV) and small cell variant. The large-cell variant is the vegetative form of the bacteria seen in infected cells. The small-cell variant (SCV) may be metabolically inactive and is the extracellular and presumably with infectious form of the organism. The SCV form of C. burnetiiis likely to be long-lived in the environment because of its resistance to osmotic stress, physical disruption, and chemical agents. Two phases of the bacterium have been described: the highly virulent phase I organisms are found in the infected hosts and insect vectors. The phase II organisms are less virulent or devoid of virulence for mammalian hosts and are obtained through multiple passages of chicken embryos.
Coxiella burnetii is a potential bioterrorism and occupational hazardous agent. Q fever has been described worldwide except in Antarctica and New Zealand. Through the air-borne, it can be inhaled by humans. A single organism of C. burnetii may cause disease in a susceptible person. In animals, C. burnetii can infect many animal species, including domestic animals, birds, reptile, wildlife, and arthropods such as ticks. Cats and dogs may represent reservoirs of C. burnetii. Dogs may be infected by tick bites, by consumption of placentas or milk from infected ruminants, and by aerosol. The possibility of human Q fever acquired from infected dogs and cats has been reported. The infected animals are generally asymptomatic, but in mammals they may induce pneumonia, abortion, stillbirth, and delivery of weak lambs, calves or kids. The Coxiella burnetii-infected herds of cows have showed shedding the organisms within the milk for 13 months. The ewe can shed the organisms within the vaginal mucus for 71 days. People who may come into contact with infected animals are at the greatest risk, including farmers, slaughterhouse workers, laboratory workers, and veterinarians. In humans, C. burnetii causes highly variable clinical manifestations, ranging from acute to fatal chronic infections. However, about 60% of the human infections are asymptomatic seroconversions. Acute Q fever in humans displays mainly flu-like symptoms, atypical pneumonia or granulomatous hepatitis. Various rare clinical signs of meningoencephalitis, endocarditis, pericarditis, pancreatitis, and abortion have also been described. It is prudent for pregnant women to limit the contact with infected animals, especially with fetal fluids and unpasteurized milk.
In Taiwan, the first case of acute human C. burnetii infection was reported in 1993. Since 2005, samples have been routinely collected from goats, sheep, cattle, and wildlife for the study of the seroprevalence and histopathological changes of Q fever and the DNA sequence of C. burnetii. The results indicate that Q fever should be considered as a possible pathogen in association with the commonly observed abortion in goats, cattle, and wildlife in Taiwan. To our knowledge, this is the first diagnosis of Coxiella burnetii infection in Taiwan livestock.
Placenta, chorioallantois: Placentitis, necrotizing, subacute, multifocal, severe, with intraepithelial and intratrophoblastic coccobacilli.
The contributor offers an excellent opportunity to identify, describe, and interpret lesions in an organ not often observed in histologic section. Conference participants spent some time discussing the components of the placenta and using the identification of individual layers and their orientation to infer a more specific location of these sections. The placenta is comprised of endometrium (the maternal component) and the fused chorioallantoic membranes (CAM, the fetal component). The indeciduate nature of ruminant placentas implies the maternal and fetal components are in contact but not intimately fused. Additionally, ruminants have cotyledonary placentation, meaning there are numerous but isolated areas where the CAM contributes to the placenta. In these areas, the CAM villi insert into pockets or crypts in the area of the endometrium known as caruncles. Specific to small ruminants, the caruncles have lost their epithelium, leaving 5 tissue layers which separate maternal and fetal blood: endothelium, connective tissue, and epithelium of the CAM, and endothelium and connective tissue of the endometrium.(1) Conference participants concluded there is no maternal tissue within these sections, and the presence of amnion and the CAM villi indicates these must have been collected from the cotyledon. Additionally, there are prominent eosinophilic crystals (hemoglobin) within an area of the CAM epithelium indicating the likelihood of a junction between cotyledonary and intercotyledonary placenta, as this is the location where hemophagocytosis is often most prominent.
Although Coxiella burnettis zoonotic potential earns it the distinction of being an abortion agent of great importance, there are many other infectious causes of abortion outbreaks in ruminants which may induce similar placental lesions. Conference participants discussed other differentials for this case, including bacteria: Chlamydophila abortus, Histophilus somni, Yersinia pseudotuberculosis, Salmonella spp., Trueperella pyogenes, Leptospira spp., Listeria monocytogenes, Campylobacter spp., and Brucella spp., parasites: Toxoplasma gondii, Neospora caninum, Sarcocystis spp., and Tritrichomonas foetus, and viruses: Border disease virus, Wesselsbron, Bluetongue, Bovine herpesvirus 1, and several Bunyaviruses. Some are easily differentiated based on lesion distribution in the placenta, such as T. gondii and Y. pseudotuberculosis which both exclusively target the cotyledons.(9)
C. burnetti is known to infect many species and has recently been described as an emerging infectious disease in birds, in which it exhibits variable tissue tropism, with the most recent reports identifying myocarditis and hepatitis in a parrot.(10) Ectoparasites such as ticks are often cited as important vectors of transmission; however, intranasal inoculation was determined to be most effective in causing disease in pregnant goats.(8) Trophoblasts in the allantochorion of the placenta are the primary target of infection and replication of the bacteria; and their main excretion route from these infected goats is during delivery of the aborted fetus, as bacterial DNA was not detected in feces or vaginal mucus in a recent study.(8)
C. burnetti is considered highly infectious, though its inoculation does not necessarily translate into reproductive disease. A recent publication identified the organism in 75% of abortion submissions based on PCR, but when combined with histopathology and additional ancillary testing, C. burnetti was only determined to be the actual cause in 21% of those cases. The more commonly identified cause of abortions in this study was T. gondii, with Campylobacter spp. and Chlamydophila spp. also being more prevalent than the agent of Q fever.(4)
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