Experimental Infection of North American Sheep with Ehrlichia ruminantium

Abstract

Ehrlichia ruminantium, a tick-borne rickettsial, causes heartwater in ruminants resulting from vascular damage. Severity of heartwater varies greatly in ruminant species and breeds, age of animals and for diverse geographic E. ruminantium strains. E. ruminantium and a tick vector, Amblyomma variegatum, originating from Africa, are well established in certain Caribbean islands two centuries ago. Besides the possibility of introduction of heartwater through African exotic animal importation, presence of the pathogen, and the tick vector in the Caribbean pose a high risk to ruminants in the USA and other western hemisphere countries. Scientific evidence supporting the heartwater threat to nonendemic regions, however, is lacking. We describe the first infection study in sheep reared in the USA with seven E. ruminantium strains. All infected sheep exhibited clinical signs characteristic of subacute to subclinical disease, which included labored breathing, depression, coughing, and nasal discharges. Gross and microscopic lesions consistent with heartwater disease including edema and hemorrhage were observed in several organs. Pathogen-specific IgG antibody response was detected in animals infected with all seven strains, while molecular analysis confirmed the pathogen presence only when infected with in vitro cultures. This is the first infection study demonstrating severe heartwater in sheep reared in North America.

Keywords: Anaplasmataceae pathogens; Cowdria ruminantium; heartwater; rickettsials; tick-borne diseases.

Figure 1

Gross pathological lesions observed in White Dorper breed sheep. Tissue samples collected on day 28 post infection (challenge) were assessed in sheep infected with E. ruminantium blood stabilates. (A) Trachea, submucosal congestion and hemorrhage (arrow). (B) Lung, diffuse and severe congestion and edema. Note the copious amounts of foamy fluid oozing from the cut section (arrow). (C) Heart, pericardial serosanguineous fluid (arrow).

Figure 2

Histopathological lesions observed in White Dorper breed sheep at 28 days following E. ruminantium blood stabilate infection. (A) Lung, moderate interstitial congestion and edema. 10× magnification, H&E. Insert: Higher magnification of a bronchiole filled with edema fluid. (B) Trachea, severe lymphoplasmacytic tracheitis (arrow) with severe edema (asterisk). 20× magnification, H&E. (C) Heart, mild multifocal lymphoplasmacytic myocarditis (arrowhead) with mild multifocal hemorrhage (black arrows). 20× magnification, H&E. (D) Heart, severe myocardial edema and hemorrhage (arrows). 10× magnification, H&E. (E) Aorta, tunica media edema (asterisk) and adventitial hemorrhage (arrow). The lumen of the vessel is marked with the letter “L”. 2× magnification, H&E. Insert: Higher magnification of the blood vessel wall edema (asterisk). (F,G) The two panels depict mild (F) to moderate (G) cortical cerebral edema (arrows). 10× magnification, H&E. Insert: Higher magnification of a cerebral meningeal vessel (asterisk) with lymphoplasmacytic perivascular infiltrates (arrowhead).

Figure 3

E. ruminantium-specific IgG response in sheep infected with blood stabilates. Antigen-specific IgG antibodies were measured in the plasma samples collected at day zero (prior to infection) and multiple time points post infection challenge by ELISA analysis. Average absorbance values of plasma collected from each sheep at each time point assessed in triplicate wells were plotted against the blood sampling days. Whole cell lysate prepared from culture-derived purified E. ruminantium Crystal Springs strain was used to coat the plates.

Figure 4

Gross pathological lesions observed in sheep infected with in vitro cultured E. ruminantium strains. (A) Trachea: Mild petechial submucosal hemorrhage (arrow). (B) Lung: Severe and diffuse congestion and edema. Note the foamy serosanguineous fluid oozing from the cut section of lung (orange arrows). (C) Heart: Subepicardial petechial hemorrhage (arrows). (D) Cross section of a congested and hemorrhagic supramammary lymph node (dotted line and arrow). (E) Enlarged and hemorrhagic mediastinal lymph node.

Figure 5

Histopathological lesions observed in sheep infected with in vitro cultured E. ruminantium strains. (A) Lung: Moderate interstitial congestion and edema. 4× magnification, H&E. Insert: Higher magnification of a bronchiole with edema fluid and several foamy macrophages (arrowhead). (B) Lung: Perivascular (arrows) to interstitial lymphoplasmacytic pneumonia (lumen of blood vessel marked with asterisk). 40× magnification, H&E. (C) Trachea: Submucosal hemorrhage (asterisk) with lymphoplasmacytic tracheitis. Arrow points to inflammation extending deeper in the submucosa, around submucosal glands. 10× magnification, H&E. (D) Heart: Moderate multifocal lymphoplasmacytic myocarditis (arrows). 40× magnification, H&E. (E) Aorta: Moderate vascular wall edema. “L” denotes the lumen of the vessel. 2× magnification, H&E. Insert: Higher magnification of vascular wall edema. Note separation of elastic fibers by edema fluid (asterisk). (F) Lymph node: Extensive interstitial congestion and hemorrhage (arrows). 4× magnification, H&E.

Figure 6

E. ruminantium-specific IgG response in sheep infected with in vitro cultured E. ruminantium strains. Antigen-specific IgG antibodies were measured in the plasma samples collected at multiple time points post infection challenge by ELISA analysis. Average absorbance values of plasma collected from each sheep at each time point assessed in triplicate wells were plotted against the blood sampling days. The day zero represents plasma samples collected from sheep prior to infection challenges. E. ruminantium Crystal Springs strain culture-derived purified antigens were coated on the ELISA plates.