EBA was historically diagnosed with a combination of unique fetal pathology, elevated levels of immunoglobulin in fetal serum, and history of the dam being grazed in areas considered endemic for the disease during pregnancy.^2,3 Gross and microscopic pathology associated with EBA is typically dramatic. Aborted fetuses are usually in excellent condition (not autolyzed) and present with a variety of gross abnormalities that can include extensive ascites with excessive fibrin (Figure 61.1a) resulting in a distended abdomen, swollen liver, enlarged spleen and lymph nodes (Figure 61.1b), skin lesions (Figure 61.1c), and petechial hemorrhages on mucosal membranes (oral cavity, tongue and eyelids) (Figure 61.1d) and in the thymus (Figure 61.2a). Microscopically, lesions are widespread and have a vascular orientation characterized by infiltration with lymphocytes and mononuclear phagocytes.^2,3 Focal necrotizing lesions may be present in organized lymphatic tissues and pathognomonic lesions occur in the thymus, characterized by depletion of cortical thymocytes and infiltration with macrophages³ (Figure 61.2b).

Microbiological diagnosis of EBA continues to be hampered by an inability to culture the causative agent. While limited short-term replication of aoEBA has been observed in primary cultures of macrophages derived from fetal lymphoid necropsy tissues, substantive propagation of the bacterium in either primary or established cell lines has not been possible; all attempts to culture aoEBA on synthetic media have also failed.⁵ Polymerase chain reaction (PCR) assays, both traditional and TaqMan-based, have been developed and target the 16S bacterial ribosomal gene.^6–8 Both assays can be used to identify the presence of aoEBA in necropsy tissues of infected fetuses presenting with classical EBA pathology. The TaqMan is the assay of choice when identifying low levels of bacteria in necropsy tissues derived from fetuses suspected of being infected, such as fetuses presenting with equivocal or nonclassical EBA pathology and weak calves that may or may not suckle. The TaqMan assay is also preferable for identifying aoEBA in the vector as only the occasional infected tick will have sufficient levels of bacteria detectable by a standard PCR.⁷

Routine bacterial histologic stains do not demonstrate the aoEBA agent in fetal tissues so a modified Steiner silver staining technique applied to the distantly related deltaproteobacterium Lawsonia intracellularis was used to microscopically visualize aoEBA in sections of formalin-fixed necropsy tissues. Steiner stain and immunohistochemistry applied to thin sections of formalin-fixed necropsy tissues (Figure 61.3a,b), and immunofluorescence applied to impression smears and/or homogenates of fresh tissue, are the current methods of choice for microscopic diagnosis of EBA.^9,10 A fluorescent antibody-based serologic assay is currently being validated. The assay appears both specific and sensitive and should prove to be a useful diagnostic when the majority of aoEBA has been eliminated by the fetal immune system. However, interpretation of fetal serology will be difficult in calves that have suckled due to the presence of maternal antibody.

Pathogenesis

The aoEBA apparently gains entrance to the developing fetus across the placenta from the infected dam. The dam, whether infected by tick bite or experimental inoculation, presents with no overt clinical signs. Reactions to experimental tick bites recorded in historical communications were most likely a response to contaminating microbes and/or tick saliva. The fetus has a “window” in development in which it is susceptible to an infection that will ultimately lead to fetal disease. This window is poorly defined but susceptibility is generally believed to be 60–140 days of gestation. Susceptibility early in gestation (<60 days) is not known. While the pathogen does not appear to impact conception, the possibility that it may induce early embryonic death in a small percentage of infected animals is under investigation. Infection of the fetus is a slow process, with definable lesions not becoming evident for 2–3 months.³ The bacterium appears to replicate intracellularly in histiocytes as determined by immunohistochemistry (Figure 61.2); replication is extremely slow (~1 day) when observed in short-term cultures of macrophages derived from fetal necropsy tissues. The intracellular nature of the infection, vascular orientation of lesions throughout the fetus, and slow and persistent development of disease would all be consistent with the hypothesis that lesions are immunologically mediated. Slow growth of the bacteria is undoubtedly responsible for slow dissemination throughout the body and lesion development coinciding with development of immunologic maturation of the fetal immune system.

Pregnant sheep do not appear to be susceptible to foothill abortion; unpublished studies in which pregnant ewes were infected in the first trimester resulted in the birth of healthy lambs. These latter studies would be consistent with the hypothesis that the bacteria replicate and disseminate slowly, and in the case of sheep the gestational period is too short to develop immunologically mediated lesions and abortion. Mule deer (Odocoileus hemionus) serve as an important host to the tick vector, but there is no evidence of EBA occurring in this species. Their gestation period is intermediate between sheep and cattle (6–7 months) and may be too short to allow for disease development. Alternatively, regular exposure to the organism through tick bites may confer immunity and, in those instances when abortions occur, fetuses would be rapidly scavenged leaving no evidence. Multiple attempts to produce disease in immunocompetent laboratory animals, such as mice and rabbits, have failed.

Mice with severe combined immunodeficiency disease (SCID) are susceptible to infection with aoEBA. C3H-scid mice develop evidence of wasting at 7–8 weeks following infection and require euthanasia 2–3 weeks later.¹⁰ The only obvious gross lesion is an enlarged spleen that harbors large numbers of infectious bacteria. Cryopreserved aoEBA-infected SCID mouse spleen cells are an excellent source of bacteria to predictably transmit infection to pregnant heifers. Unlike bacteria derived from the bovine fetus, recovered organisms are not opsonized and appear to have excellent viability. The absence of EBA-like lesions in immunodeficient infected mice would support the hypothesis that the fetal bovine lesions are immunologically mediated as suggested above.¹⁰

Transmission

Observations that the distribution of the Ornithodoros coriaceus tick could be superimposed upon the distribution of foothill abortion precipitated studies to establish them as potential biological vectors.^4,7,8 This unusual soft-shelled tick (commonly called the Pajaroello tick) carries a colorful past as Native Americans in California feared its bite over that of a rattlesnake. They believed a second or third exposure to this tick bite would result in death. The tremendous pain and swelling of appendages that sometimes occurs following sequential Pajaroello tick bites is assumed to be an extreme allergic reaction to the tick saliva and/or associated contaminating microbes. An unusual and classical experiment was conducted in which naive pregnant heifers were divided into two groups in an area considered endemic for the disease. One half of the heifers were maintained in tree houses with the tree bases tightly wrapped with metal to interfere with the potential ascent of crawling insects, and the other half were maintained on the ground below. All tree-housed heifers produced healthy calves while foothill abortion occurred in the land-based animals. Studies were subsequently conducted in which wild-trapped ticks were experimentally fed on pregnant naive heifers, further demonstrating disease transmission.^4,5 The O. coriaceus tick has been collected in Mexico, California, Nevada, and Oregon (Figure 61.4), but foothill abortion has only been described in the above-mentioned three US states.^7,8,11 Cattle ranching practices in Mexico may well be responsible for a lack of diagnosis of foothill abortion in this country; aborted fetuses can be rapidly scavenged in free-ranging cattle herds, making diagnosis unlikely.