Concurrent Infection with <i>Anaplasma phagocytophilum</i> and <i>Mycoplasma haemolamae</i> in a Young Alpaca

Abstract

A 7-month-old 30-kg male alpaca was referred to the large animal hospital at Tufts University Cummings School of Veterinary Medicine in November for evaluation of partial anorexia, mild colic, ataxia progressing to recumbency, and suspected blindness of 1-day duration. Upon presentation, the alpaca was anxious, recumbent, and unable to support its head in an upright position. Physical examination indicated normothermia (100.3°F; reference range, 99.5–101.5°F), mild tachycardia (90 beats/min; reference range, 50–80 beats/min), and tachypnea (40 breaths/min; reference range, 10–30 breaths/min) with increased respiratory effort, cold extremities, and dry mucous membranes. A regular heart rhythm was present, and normal bronchovesicular sounds were auscultated over both lung fields. Auscultation of the abdomen showed reduced intestinal borborygmi. Other findings on physical examination included bilateral mydriasis with lack of menace and sluggish direct and consensual pupillary light responses (PLR). Motor and sensory function was present in all 4 limbs. Venous blood gas analysisa indicated acidemia (pH 7.280; reference range, 7.337–7.467) associated with metabolic acidosis (serum bicarbonate concentration 11 mmol/L; reference range, 20–26 mmol/L), hyperlactatemia (14.2 mmol/L; reference range, <2 mmol/L), and marked hypoglycemia (<20 mg/dL; reference range, 98–133 mg/dL), which was confirmed on repeated whole blood analysis. Plasma electrolyte concentrationsa were within normal limits. Immediate resuscitation therapy included Plasma-Lyteb with 5% dextrosec (20 mL/kg bolus) followed by hetastarchd (10 mL/kg IV). Crystalloid fluid therapy was continued using 20 mL/kg IV boluses until general endpoints of resuscitation (eg, urination, central venous pressure 5–8 mmHg and decreased serum lactate concentration) were achieved. Thiaminee (20 mg/kg IM) was administered to combat potential poliencephalomalacia. Short-term nasal oxygen therapy (5 L/min) was initiated until respiratory disease could be ruled out. Abnormalities on CBC included leukocytosis (19.1 × 103 cells/μL; reference range, 6.1–17.1 × 103 cells/μL) with neutrophilia (17.4 × 103 cells/μL; reference range, 3.4–12.7 × 103 cells/μL). Numerous small (<1 μm), epicellular, basophilic, and coccoid to ring-shaped structures consistent in appearance with Mycoplasma haemolamae were present on the erythrocytes (Fig 1). In addition, occasional neutrophils and rare eosinophils contained clusters of small basophilic coccoid structures consistent in appearance with Anaplasma phagocytophilum morulae (Fig 1). Serum biochemical abnormalities were limited to marked hypoglycemia (<2 mg/dL; reference range, 98–133 mg/dL) and hypoproteinemia (5.2 g/dL; reference range, 5.5–7.5 g/dL) with mild hypoglobulinemia (1.7 g/dL; reference range, 1.9–3.6 g/dL). Photomicrograph, Anaplasma phagocytophilum morulae within neutrophil (white arrow), Mycoplasma haemollama surrounding erythrocytes (black arrows). Scale bar = 5 μm, Wright's stain. Possible causes for the presenting neurological abnormalities included Eastern equine encephalitis (EEE), West Nile virus (WNV), equine herpesvirus-1 (EHV-1), bacterial meningitis, polioencephalomalacia, listeriosis, brain abscess, trauma, and paralaphostrongylosis. Lumbosacral cerebral spinal fluid (CSF) was submitted for cytology, quantification of IgM against WNV, and plaque-reduction-neutralization test to identify EEE antibodies.f These tests have been used previously in assessment of WNV and EEE antibody response to vaccination in llamas and alpacas.1,2 Serological testing for EHV-1 was performed by serum neutralization on whole blood.g The CSF was transparent and colorless with normal protein (39 mg/dL; reference range, 31–67 mg/dL), cytological findings (total nucleated cells 1/μL; reference range, 0–3/μL), and cell differential (58% mononuclear cells, 42% lymphocytes), which eliminated a septic process. Furthermore, all test results for EHV-1, EEE virus and WNV were negative, making viral exposure unlikely. Primary or secondary gastrointestinal disease also was considered, based on the animal's history of transient colic and anorexia. Abdominal ultrasound examination showed decreased intestinal motility and mild small intestinal distension (diameter, 1.4–1.8 cm; reference range, 1.2–1.6 cm),3 however, intestinal wall thickness was within normal limits (reference range, 0.2–0.3 cm).3 Abdominal fluid analysis was unremarkable (total nucleated cell count 0.563 × 103/μL, reference range, <3 × 103/μL; total protein, <2 g/dL, reference range, <2.5 g/dL),4 and thus did not suggest clinically relevant vascular compromise of the gastrointestinal tract. Formed feces were present and fecal flotation analysis by centrifugation showed few Eimeria sp. ova, but was otherwise unremarkable. Based on the hematologic findings above, continued therapy was directed toward treatment for Mycoplasma and Anaplasma infection, gastrointestinal disease, possible thiamine deficiency, and metabolic acidosis. Medications administered included oxytetracycline hydrochlorideh (10 mg/kg IV in 250 mL 0.9% NaCl q12h) for treatment of both Mycoplasma and Anaplasma infection, enrofloxacini (10 mg/kg SC q24h) for potential Escherichia. coli enterocolitis, thiamine (20 mg/kg IM q12h), amprolium hydrochloride j (10 mg/kg PO q24h for 5 days) in the event that Eimeria infection contributed to the gastrointestinal abnormalities, and flunixin megluminek (1.1 mg/kg IV q12h for 2 days) for analgesic, anti-inflammatory, and antiendotoxic properties. In addition, Plasma-Lyteb infusion was continued and ranged from 60–120 mL/kg/d, depending on improvement of cardiovascular parameters. Within the first 4 hours of hospitalization, the patient became mildly febrile (T=102.8°F; reference range, 99–101.5°F) and only demonstrated slight improvement in blood lactate concentration (10.8 mmol/L) in the face of decreasing PCV (17%; reference range, 25–40%) and serum protein concentration (4.4 g/dL). Appropriate urination and low urine specific gravity (1.014) suggested adequate rehydration. Proteinuria was not observed. A measurement of colloid oncotic pressure obtained at that time was only mildly decreased (18.3 mmHg; reference range, 20.3 ± 1.95 mmHg) and additional colloids were not administered. The initial PCV and total protein concentration may have been artificially increased secondary to hemoconcentration. Colloid administration may have further decreased relative serum protein concentration. Because of the intermittent signs of agitation and distress during handling, butorphanoll (0.2 mg/kg IM) was administered as a mild sedative and analgesic, and the animal was placed in a quiet, isolated stall with limited stimuli. Heart rate and rhythm were continuously monitored by ECG. The alpaca remained dull and recumbent, but over the next several hours vital signs continued to stabilize. Twelve hours after initial presentation the alpaca remained quiet and mildly febrile (102.4°F) but was able to stand unassisted. Over the next 12 hours, its attitude continued to improve, and it urinated and defecated normally. No other signs of agitation or discomfort were noted. Thoracic radiographs obtained after the animal's condition had stabilized disclosed a mild interstitial pattern, but were otherwise unremarkable. IV fluid support was discontinued 48 hours after presentation (day 3 of hospitalization), but mild anemia (PCV, 19–24%) and hypoproteinemia (4.6–5.2 g/dL) persisted throughout the animal's hospitalization. On day 2 of hospitalization, the condition of the alpaca had improved sufficiently to perform a more thorough neurological evaluation. Although the animal remained moderately weak with transient muscle fasciculations, no gait or conscious proprioceptive abnormalities were detected. The menace response remained absent, but PLR were normal in both eyes by day 2. A fundic examination also was normal bilaterally, supporting central (cortical) blindness. The transiently sluggish PLR upon presentation may have occurred secondary to decreased sensory input, hypoglycemia, or overriding sympathetic stimuli. After 5 days of hospitalization, hematologic evaluation of the alpaca was normal, and neither A. phagocytophilum nor M. haemolamae were detected on blood smears. IV oxytetracycline and enrofloxacin were discontinued 7 days after presentation and the animal was discharged on long-acting oxytetracyclinel (20 mg/kg SQ q48h) and thiamine (10 mg/kg IM q12h). Follow-up examination 2 weeks after discharge showed normal menace, PLR and fundic examination indicating resolution of the blindness. Hematologic findings were unremarkable at this time. Convalescent titers to A. phagocytophilum were not obtained. To the authors' knowledge, this case is the first report of concurrent M. haemolamae and A. phagocytophilum infection leading to neurological, cardiovascular, and gastrointestinal signs in an alpaca. M. haemolamae belongs to a family of small extracellular bacteria that cause hemoplasmosis in a variety of species including camelids.5–7 The mode of transmission of M. haemolamae in camelids is unknown, but biting or blood sucking vectors such as ticks are highly suspected.8 The mechanism by which mycoplasma species cause hemolytic anemia is thought to involve an immune-mediated process in response to bacterial attachment to and deformation of the outer surface of red blood cells.7,9 In healthy camelids, these bacteria occasionally are an incidental finding and a subclinical carrier state may exist. Ill or otherwise immunocompromised individuals often present with mild to severe anemia. In herds with high seroprevalence, animals may present acutely with recumbency and weakness, or may demonstrate chronic illness including weight loss and decreased fertility.6,9 In this alpaca, the presence of M. haemolamae in the peripheral blood was confirmed by polymerase chain reaction (PCR) of the 16S ribosomal RNA genem and most likely represented an opportunistic infection. This alpaca's relatively mild anemia in the face of a very high parasite load suggested an acute onset of disease. Granuocytic ehrlichiosis is caused by A. phagocytophilum (formerly Ehrlichia phagocytophila), a tick-borne obligate intracellular bacterium. In the northeastern United States Ixodes scapularis is the proposed vector.10 Clinical signs of disease may vary and include fever, lethargy, anorexia, and ataxia, whereas typical laboratory abnormalities include lymphopenia, anemia, and thrombocytopenia. Intracytoplasmic inclusions (morulae) in neutrophils may be observed, but these inclusions are not always present in clinically affected animals. In the current report, PCR and 16S ribosomal RNA gene sequencing of an anticoagulated blood samplen,o confirmed infection with A. phagocytophilum. Antibody titers for A. phagocytophilump upon admission were negative at a dilution of <1 : 10. Titers are considered positive at a dilution of ≥ 1 : 50.q To date, a single r case report previously documented ehrlichial infection in a llama11 from California. PCR amplification and nucleotide sequencing of the 16s RNA gene of that organism identified it as being closely related to the Ehrlichia (now A. phagocytophilum) genogroup. Clinical signs and hematologic abnormalities in the llama were similar to those noted in other species. The alpaca described in the current report represents the first confirmed documentation of A. phagocytophilum infection of an alpaca and the first documented ehrlichial infection of a camelid in the northeastern United States. The observed clinical signs of ataxia, anorexia, lethargy, and fever along with A. phagocytophilum inclusions in this alpaca are similar to those reported in the llama, and also are typical of other affected species. The absence of fever upon presentation was unusual, but the animal was profoundly hypoglycemic, which may have explained its hypothermia. This case also was unique in that several additional clinical abnormalities occurred, including blindness, profound hypoglycemia and hyperlactatemia, suggesting the possibility of multiple disease processes in the face of concurrent M. haemolamae infection. The cause of central blindness in the alpaca of this report remains speculative. Polioencephalomalacia can lead to cortical blindness that may persist several weeks to months in animals even after resolution of underlying clinical abnormalities.12,13 In this alpaca, severe metabolic derangements also may have contributed to impaired cortical function. Acute blindness secondary to monocytic or granulocytic ehrlichiosis has been reported in dogs,14 but ocular abnormalities including uveitis, retinal detachment, and intraocular hemorrhage are typical. These findings were not observed in the alpaca of the current report. Furthermore, neurological signs in horses infected with A. phagocytophilum may be related to focal CNS lesions secondary to localized vasculitis.15 Localized vasculitis thus may have affected the visual cortex and contributed to central blindness in our patient. Differential diagnoses for the observed hypoglycemia in this alpaca included prolonged starvation, impaired gluconeongenesis (eg, hepatic disease), increased use of or impaired metabolism of glucose (sepsis), specific parasitic infection (M. haemolamae), or measurement artifact. Physical examination and hematologic analyses eliminated the presence of starvation, hepatic disease, and acute sepsis. Because hypoglycemia was identified both on blood gas analysis and stall-side use of a hand held glucometer,k a measuring artifact was considered unlikely. Hypoglycemia in association with mycoplasma infection has been observed in several species, including camelids.5,6,16,17 Parasitic consumption of glucose may exceed host production or host red blood cell consumption may exceed production.5,6,16,17 Given the high parasite load detected on the alpaca's blood smear, the observed hypoglycemia may have been secondary to M. haemolamae infection. Several factors could account for the markedly increased lactate concentration noted in this alpaca, including inadequate tissue perfusion (hypovolemia), endotoxemia, systemic hypermetabolism, relative thiamine deficiency as well as increased lactate release from the gastrointestinal tract. Concurrent decreases in serum protein concentration suggest gastrointestinal protein losses. This finding, in addition to the alpaca's signs of colic, support an underlying gastrointestinal abnormality such as colitis or enteritis. The Eimeria sp. ova noted on fecal floatation also may have represented a clinically relevant degree of intestinal parasitism. Finally, severe mycoplasma infection alone or in combination with disease from A. phagocytophilum may have further contributed to increased lactate concentration in this alpaca. Humans with malaria and dogs with babesiosis both can experience severe derangements in carbohydrate metabolism that present as profound hypoglycemia and hyperlactatemia.18–21 Additionally, mycoplasma infection is thought not only to cause hypoglycemia, but also to induce increased lactate production by red blood cells,16 possibly due to parasite glucose metabolism, although the clinical importance of this effect is unknown. The alpaca described in the current report was part of a small, well-managed herd. No other animals in the herd had signs of illness at the time the alpaca presented for referral. The owner had lost 2 animals in the previous several months to acute onset of disease of unknown origin and had commented that ticks frequently were observed on the animals. Horses on several farms in the area had documented positive titers for A. phagocytophilum, with a few horses having signs of clinical disease. The overall seroprevalence of A. phagocytophilum in the resident alpaca's herd, evaluated shortly after patient discharge, was 38% with 23% positive at >1 : 320. CBC and serum biochemistry profiles performed on these animals all were within normal limits, and no abnormalities were detected on blood smear. In summary, this report represents the first case of A. phagocytophilum infection in an alpaca, associated with herd exposure, in the northeastern United States. Concurrent infection with A. phagocytophilum and M. haemolamae contributed to a unique clinical presentation in this animal and a common tick vector could not be ruled out. Infection with A. phagocytophilum in alpacas may mimic the presentation in other species, and can be associated with severe lethargy, ataxia, anorexia, fever, and cardiovascular compromise. It should be considered as a differential diagnosis in the face of acute, infectious, unspecific critical illness in young, and adult camelids. A. phagocytophilum exposure of alpacas most likely is common in endemic areas, but clinical illness may only be observed in a small number of exposed animals. aStat Profile, Critical Care Xpress, Nova Biomedical, Waltham, MA bBaxter Health Care, Deerfield, IL cHospira Inc, Lake Forest, IL dHospira Inc eThiamine Hydrochloride, Butler Animal Health, Dublin, OH fMassachusetts State Laboratory Institute, Jamaica Plain, MA gAnimal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY hOxibiotic 100, IVX Animal Health Inc, St Joseph, MO iBaytril 100, Bayer Health Care, Shawnee Mission, KS jAmprolium hydrochloride, Duluth, GA kFlunixamine, Fort Dodge Animal Health, Fort Dodge, IA lFort Dodge Animal Heath mPathobiology Diagnostic Services, Auburn University, Auburn, AL nVector Borne Diagnostic Laboratory, North Carolina State University oLucy Whittier Molecular and Diagnostic Core Facility, University of California at Davis pWILD2 Ehrlichia equi immunofluorescence, IDEXX Laboratories, North Grafton, MA qAscensia Elite XL, Mishawaka, IN rLA 200, Phizer Animal Health, Exton, PA