Naturally Occurring Ehrlichia ewingii and Mycoplasma sp. Co-Infection in a Goat.

Anaplasma

alkaline phosphatase

aspartate aminotransferase

base pairs

creatinine kinase

celsius

deoxyribonucleic acid

Ehrlichia

fahrenheit

gamma‐glutamyl transferase

glyceraldehyde‐3‐phosphate dehydrogenase

kilogram

mean cell volume

mean corpuscular hemoglobin concentration

Mycoplasma

polymerase chain reaction

ribosomal ribonucleic acid

A 9‐year‐old nonpregnant, nonlactating doe Boer goat was examined because of a 2‐day history of not being able to stand. Other than diarrhea associated with coccidiosis in the first year of life, the goat did not have a history of illness. The owner had obtained the goat at approximately 2 months of age. The goat lived with 3 other goats in the same pen on the same premise located on the coastal plains of North Carolina. The goats spent the majority of time in a barn, with access to a wooded 1‐acre lot where they browsed. Routine deworming prophylaxis was verified by fecal egg counts. The goat was vaccinated for clostridial diseases, was fed 1 cup of 13.5% protein commercial goat pellets twice a day, and had free access to good quality coastal Bermuda hay.

At presentation for recumbency, the goat was nonweight bearing on the right forelimb and could stand only with assistance, but was unable to walk. Otherwise, the goat was bright, alert, responsive, and had a good appetite. Body condition score (5/5), body weight (65.5 kg), rectal temperature (39.2°C [102.5°F]), heart rate (80 beats per minute), respiratory rate (24 breaths per minute), mucous membrane color, and capillary refill time were normal. An abscess was present on the ventral aspect of the mammary gland.

To further assess the lameness, lateral, craniocaudal, and oblique radiographs of the right humerus were obtained,1 and a mildly comminuted, moderately proximo‐caudally and medially displaced short oblique fracture of the proximal humeral diaphysis was identified (Fig 1). Marked soft tissue swelling was associated with the fracture. Mild rounding and blunting of the fracture margins without evidence of callus formation were observed and, consequently, some degree of chronicity (>7–10 days) was considered likely.1

Figure 1

Craniocaudal (A) and lateral (B) radiographs of the right humerus of a 9‐year‐old female Boer goat presenting with nonweight bearing lameness. Radiographs show an oblique fracture of the proximal humeral diaphysis that is associated with marked soft tissue swelling. Very mild rounding and blunting of the fracture margins (arrows) without evidence of callus formation are consistent with a fracture of at least 7–10 days duration.

A CBC2 identified mild macrocytic normochromic anemia (PCV, 20%; reference range, 22–38%; mean cell volume [MCV], 26.4 fL; reference range, 16–24 fL; mean corpuscular hemoglobin concentration [MCHC], 34.3 d/dL; reference range, 30–37 g/dL). The total leukocyte count was 9,900/μL (reference range, 5,100–17,200/μL)2 and characterized by a mild regenerative left shift (segmented neutrophils, 8,800/uL; reference range, 1,100–8,900/μL; band neutrophils, 100/μL; reference range, 0/μL),2 mild lymphopenia (lymphocytes, 900/μL; reference range, 1,200–10,500/μl)2 and normal numbers of monocytes (100/μL; reference range, 0–300/μl).2 Microscopic evaluation of a blood smear disclosed moderate anisocytosis, mild macrocytosis, and occasional basophilic stippling, suggestive of a regenerative response. Occasional eccentrocytes indicated oxidative damage, potentially related to the inflammatory illness. Approximately 1–2% of the segmented neutrophils contained single, approximately 2–3 × 2–3 μm, tightly packed, basophilic granular clusters of organisms within the cytoplasm, consistent with morulae (Fig 2). No other etiologic agents were observed. Platelet number estimate on the blood smear appeared adequate. Serum biochemistry abnormalities3 included mildly increased activities of AST (227 IU/L; reference range, 35.5–72.0 IU/L2), CK (788 IU/L; reference range, 24.5–98 IU/L2), and GGT (52 IU/L; reference range, 16.0–45 IU/L2) and decreased ALP activity (35 IU/L; reference range, 77–883 IU/L2).

Figure 2

Blood smear from a 9‐year‐old female Boer goat. Mildly decreased RBC density, moderate anisocytosis, and mild macrocytosis indicate mild, likely regenerative anemia. The segmented neutrophil in the center (insert with higher magnification) contains a tightly packed, basophilic granular cluster of organisms (morula) in the cytoplasm, which was identified as Ehrlichia ewingii by polymerase chain reaction. No other etiologic agents are observed. Wright–Giemsa stain; bar = 10 μm; large image: 1,000× magnification field.

The owners elected euthanasia because of the poor prognosis associated with the humeral fracture, and declined necropsy.

For molecular microbiological identification of granulocytic morulae, EDTA anti‐coagulated blood was submitted to the Vector Borne Disease Diagnostic Laboratory at North Carolina State University College of Veterinary Medicine (VBDDL NCSU‐CVM). In addition, a comprehensive polymerase chain reaction (PCR) panel, which includes detection of Anaplasma (A.)/Ehrlichia (E.) spp., Babesia spp., Bartonella spp., Mycoplasma (M.) spp., and Rickettsia spp. was performed. Briefly, DNA was extracted from 200 μL of whole blood.4 Negative extraction controls consisting of uninfected dog EDTA‐whole blood were included. The absence of PCR inhibitors was demonstrated by the amplification of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH).3 The PCR conditions were previously described to amplify a 700‐base pair (bp) fragment of the Mycoplasma 16S rRNA gene, a 430‐bp fragment of the Anaplasma/Ehrlichia 16S rRNA gene, a 620‐bp fragment of the Anaplasma/Ehrlichia GroEL gene, and a 304‐bp fragment of the Ehrlichia sodB gene.4, 5, 6, 7 Both the Anaplasma/Ehrlichia 16S rRNA and GroEL assays were run with a modified annealing temperature of 60°C. Positive (Candidatus Mycoplasma haematoparvum, Anaplasma platys or Ehrlichia chaffeensis plasmid DNA) and negative (RNAse‐free, molecular grade water, and a DNA extraction control) were included as controls in each assay. The PCR product visualization was performed using gel electrophoresis. Amplified DNA was sequenced directly,5 and alignments were compared with GenBank sequences.6 The PCR results from the comprehensive panel yielded positive amplicons for Anaplasma/Ehrlichia 16S rRNA and Mycoplasma 16S rRNA (Table 1). Additional PCR assays (GroEL and sodB) were performed to confirm the original Ehrlichia 16S rRNA results. Amplicons were sequenced and comparisons performed using BLAST against the GenBank database. Sequence identities for the partial Mycoplasma 16S rRNA, Ehrlichia 16S rRNA, GroEL, and sodB genes with respective GenBank accession numbers are listed in Table 2, all with 100% coverage. Additional Ehrlichia species‐specific assays included Ehrlichia canis p30, E. chaffeensis sodB, Panola Mountain Ehrlichia sp. sodB and gltA, and Ehrlichia muris dsb, and did not amplify DNA (Table 2). Based on these PCR results, co‐infection with Ehrlichia ewingii and Mycoplasma sp. was diagnosed in this goat.

Table 1

Peripheral blood polymerase chain reaction (PCR) panel results from a 9‐year‐old female Boer goat with a fracture of the right humerus and morulae identified within neutrophils on the CBC

Genus PCR Target	PCR Results
Anaplasma/Ehrlichia	Positive
Babesia	Negative
Bartonella	Negative
Mycoplasma	Positive
Rickettsia	Negative

Table 2

Sequence identities for Mycoplasma 16S rRNA and Ehrlichia polymerase chain reaction (PCR) amplicons

Gene Target	GenBank Sequence Comparisons
Mycoplasma 16S rRNA	100% (552 bp) identical to an uncultured Mycoplasma spp. 16S rRNA from a white‐tailed deer in North Carolina (KC512404)4
Ehrlichia 16S rRNA	99% identical (366/368 bp) to E. ewingii, genotype Panola Mountain partial 16S rRNA gene from Amblyomma americanum (DQ365880) and E. ewingii, strain 95E9‐TS from a dog from North Carolina with granulocytic ehrlichiosis (U96436.1)
Ehrlichia GroEL	100% identical (584 bp) to E. ewingii partial GroEL gene from a naturally infected human patient with ehrlichiosis (AF195273)
Ehrlichia sodB	100% identical (303 bp) to E. ewingii partial sodB gene from a naturally infected dog (KC778986)
E. canis p30	N/A
E. chaffeensis sodB	N/A
Panola Mountain Ehrlichia sp. sodB and gltA	N/A
E. muris dsb	N/A

bp, base pairs; E., Ehrlichia; N/A, not applicable, as no PCR amplicon was obtained for DNA sequencing.

Sequences were compared to the GenBank database using the Basic Local Alignment Search Tool.

To the authors' knowledge, ours is the first report describing naturally occurring E. ewingii infection in a goat that was also co‐infected with a hemotropic Mycoplasma sp. Ehrlichia spp. are composed of a group of obligate intracellular bacteria that have a tropism for leukocytes. Ehrlichia ewingii is the causative agent of granulocytic ehrlichiosis in dogs and human ewingii ehrlichiosis.8, 9, 10 Based on serology using Ehrlichia sp.‐specific peptides, E. ewingii is the most prevalent Ehrlichia spp. that infects dogs in the south central, south eastern and mid‐atlantic United States.11 This finding is consistent with the geographic distribution of the primary vector of E. ewingii, Amblyomma americanum, the lone star tick.12 Wildlife, particularly the white‐tailed deer, are a major reservoir host for the maintenance of several A. americanum‐associated pathogens including E. ewingii.12, 13 Among domestic animals, dogs also may serve as a reservoir host for A. americanum because there is evidence of a chronic E. ewingii carrier status after acute infection.12, 14

Various Anaplasma and Ehrlichia spp. can infect domestic goats. Anaplasma phagocytophilum (referred to as tick‐borne fever in Europe) and Ehrlichia ruminantium (heartwater disease, historically the organism was designated Cowdria ruminantium) are well described, clinically and economically relevant tick‐transmitted diseases in Europe and Africa, respectively.15, 16, 17, 18 The importance of other Ehrlichia spp. in goats is less clear. Ehrlichia canis and Neorickettsia risticii (formerly Ehrlichia risticii) were shown to infect goats by inoculation of infected blood.19 The recently discovered Panola Mountain Ehrlichia sp. was transmitted to goats by infected A. americanum ticks, and natural infection of goats with E. chaffeensis also has been reported.20, 21

Domestic goats serve as hosts for all life stages of A. americanum,22 and experimental infection of goats by E. ewingii‐infected A. americanum ticks was demonstrated in 3 goats.20 However, to the authors' knowledge, natural infection of goats with E. ewingii has not been reported previously. Clinical abnormalities in the experimentally E. ewingii‐infected goats included mild pyrexia, lethargy, inappetence, serous nasal discharge, lameness, and coughing.20 None of these signs were noted in the goat of our report. Similar to our case, subclinical E. ewingii infection also has been reported in dogs.23 However, laboratory findings reported in this goat, including lymphopenia, neutrophilic left shift, decreased ALP activity, and increased GGT activity are changes reported in association with experimental E. ewingii and natural A. phagocytophilum infections in goats.15, 16, 17, 20 Decreased ALP activity in goats with Ehrlichia spp. infection is a consistent and pathophysiologically interesting observation. It has been postulated that the activity of ALP, a zinc‐dependent enzyme, is correlated with the plasma concentrations of zinc, which decrease during rickettsemic episodes as a result of the release of endogenous pyrogens and acute phase mediators.15, 17 The clinical relevance of mildly increased GGT activity in this goat is unclear because no other laboratory data indicated cholestasis. The activity of SDH would have been a sensitive marker to screen for hepatic disease, but was not determined in this goat. Increased GGT activity also was related to altered hepatic metabolic activities associated with A. phagocytophilum infection in goats,15 and increased serum transaminase activities including GGT occur in association with E. ewingii ehrlichiosis in humans.10

The mode, timing, and duration of E. ewingii infection in this goat remains speculative. Morulae were visualized in the fall when Amblyomma americanum ticks are still active in North Carolina.12 Findings of neutrophilic morulae in blood smears occurs during the acute infection, or when a persistently infected reservoir host is severely stressed or treated with immunosuppressive drugs. Underlying immunosuppression is a known risk factor for E. ewingii infection in humans,10 but it is unclear if immunosuppression might have contributed to E. ewingii infection in this goat.

Another interesting aspect of this case was the co‐infection with hemotropic Mycoplasma sp., which was not visualized on the blood smear. The amplified partial Mycoplasma 16S rRNA sequence was recently described to occur with high incidence in asymptomatic white‐tailed deer in North Carolina.4 This finding suggests that white‐tailed deer could be a reservoir host for both infections documented in this case, E. ewingii and Mycoplasma sp.4, 12, 13 Perhaps both organisms were transmitted by the same vector (A. americanum), and access to the wooded lot likely exposed the goat to infected ticks. Similar to the E. ewingii infection, the clinical relevance of the Mycoplasma sp infection is not clear in the present case. Although blood smear evaluation has a low sensitivity for detection of hemotropic Mycoplasma infection, failure to visualize organisms on the blood smear in this case suggests a low infectious burden in a chronically infected goat.24

To the authors' knowledge, this case represents the first reported case of natural E. ewingii infection in a goat that was also co‐infected with a hemotropic Mycoplasma sp., acquired in south eastern North America. The clinical relevance of naturally occurring E. ewingii infection in goats should be determined, and whether the goat is an incidental or a primary host should also be investigated. The prevalence of ticks infected with E. ewingii is increasing markedly in North Carolina,25 and E. ewingii can infect wildlife species, domestic animals, and also humans.10 Therefore, farm workers and owners of pet goats should be aware that pastures might contain infected ticks. Serological and molecular surveys of this pathogen in goats should be performed to identify the role of goats as a potential reservoir host and to determine the prevalence of infection in this species.