Premature parturition, edema, and ascites in an alpaca infected with Anaplasma phagocytophilum

Abstract

An 8-year-old alpaca was presented for fever, anorexia, edema, ascites, and premature parturition. She was determined to have Anaplasma phagocytophilum infection based on positive blood polymerase chain reaction (PCR) and positive acute and convalescent serum titers. Antibiotics and supportive therapies were administered and the alpaca made a complete recovery.

Anaplasma phagocytophilum is an obligate, intracytoplasmic coccus whose outer cell wall structure resembles that of Gram-negative bacteria. It was formerly classified as Ehrlichia equi, Ehrlichia phagocytophila, and in humans, the human granulocytic ehrlichiosis (HGE) agent (1,2). It infects granulocytes and reproduces in membrane-bound vesicles called morulae (1,2). Anaplasma phagocytophilum infects several species including horses, cattle, sheep, goats, and humans, and rarely domestic cats and camelids (2). The organism is maintained in a variety of wildlife hosts and is transmitted between animals through the bite of various species of Ixodes ticks (1). In the eastern and midwestern United States, I. scapularis has been implicated as the vector, whereas on the west coast I. pacificus is the vector. The organism is transmitted by I. ricinus in most of Europe, and by I. persulcatus in China, Japan, and Russia (2–4). Disease due to A. phagocytophilum in European domestic ruminants is also known as tick-borne fever, and has been reported in sheep, cattle, goats, and deer (2). There are no reports of infection with A. phagocytophilum in domestic ruminants in the United States. Strains infecting domestic ruminants in Europe and white-tailed deer in the United States appear to be distinct from those infecting horses, humans, and dogs based on sequences of major surface protein genes (2,5,6). The presence of high fever in animals that have been recently moved into tick-infested pastures is 1 of the first indications of tick-borne fever in sheep (7). Secondary infections have also been reported in adult cattle, along with a drop in milk yield in dairy cattle. Abortion storms may occur when non-immune pregnant ewes or cows are exposed to tick-infested pastures during the last stages of pregnancy (7).

Clinical signs presumed to be associated with A. phagocytophilum infection have been previously reported in 1 alpaca (8) and 1 llama (9). Clinical signs reported in these 2 cases and in other species are variable and include fever, lethargy, anorexia, petechial hemorrhage, edema, stiff gait, ataxia, and recumbency (3,8,9). The following case report describes previously unreported clinical signs of premature parturition, edema, and ascites in an alpaca with A. phagocytophilum infection.

Case description

An 8-year-old female Suri alpaca was presented to the Large Animal Hospital at the University of Minnesota College of Veterinary Medicine in May 2009 with her critically ill 5-hour-old cria, that was born 1 mo prematurely. The dam had previously given birth to 4 healthy full-term crias with no other abnormal reproductive history. She was vaccinated annually for tetanus, Clostridium perfringens types C and D, and dewormed monthly with ivermectin. She was also treated with albendazole once per year, given 2 wk after parturition. She had been shorn 1 wk prior to parturition and was acquired by the owner 4 mo prior to admission. She was kept on grass pasture with several other dam and cria pairs and ate free choice grass hay and 8 oz llama pellets daily. No other animals on the farm were ill. No travel of animals on or off the farm had occurred since the alpaca’s arrival at the farm 4 mo previously. On the day of parturition the alpaca had been inappetent and had decreased fecal production but no other abnormalities were reported.

On presentation, the alpaca was bright, alert, and responsive. Her temperature was elevated at 39.6°C (103.4°F); reference interval (RI): 37.2°C to 38.6°C (99°F to 101.5°F), and heart and respiratory rates were normal, 60 beats/min; RI: 50 to 80 beats/min, and 30 breaths/min; RI: 10 to 30 breaths/min, respectively (10). Mucous membranes were pink and slightly tacky, with a capillary refill time of 2 s. Based on physical examination, she was estimated to be mildly dehydrated. No cardiac murmurs or arrhythmias were ausculted, and normal breath sounds were heard over all lung fields. A few normal formed fecal pellets were produced during the examination and gastrointestinal borborygmi were decreased. Body condition score was adequate and estimated to be 5/9. Peripheral lymph nodes were not enlarged, and no lameness or ataxia was noted at the walk. Wax plugs were found in all 4 teats, which were subsequently expressed and scant milk production was noted. A mild amount of vulvar swelling without vaginal discharge was observed. The results of the remainder of the physical examination were unremarkable. As she appeared stable, the dam was given grass hay and water, and monitored while her cria was treated in the intensive care unit for symptoms associated with prematurity. Several hours later the dam’s temperature was further elevated [40.2°C (104.4°F)], but heart and respiratory rates remained within normal limits. The animal had consumed no food or water, and had passed no feces. A palpable abdominal fluid wave was detected along with abdominal distention, and muzzle and ventral edema.

Initial diagnostic tests included complete blood (cell) count (CBC), serum chemistry profile, abdominal ultrasound examination, abdominocentesis with cytological evaluation and culture of peritoneal fluid, and quantitative fecal examination. Abdominal ultrasound was performed while awaiting blood work. A significant amount of anechoic free abdominal fluid was noted, and was the only remarkable finding other than compartmental and small intestinal hypomotility. Abdominocentesis was performed, and 1 L of clear, colorless fluid was slowly drained as lactated Ringer’s solution was administered intravenously at a rate of 10 mL/kg body weight (BW) per hour to compensate for third space losses. Cytological analysis of the fluid revealed 158 nucleated cells/μL (42% large mononuclear cells, 49% non-degenerate neutrophils, 8% small lymphocytes, 1% eosinophils) and a total plasma protein of 29 g/L (reference value: < 25 g/L), consistent with a transudate (11). No organisms were noted and aerobic and anaerobic cultures of the fluid were negative.

Abnormalities in the CBC included mild anemia with a packed cell volume of 0.24 L/L (RI: 0.27 to 0.4 L/L), and slightly low hemoglobin (103 g/L; RI: 105.5 to 173.4 g/L), leukopenia (5.94 × 10⁹ cells/L; RI: 6.39 to 18.91 × 10⁹ cells/L) characterized by a low normal neutrophil count (4.51 × 10⁹ cells/L; RI: 3.25 to 13 × 10⁹ cells/L) and lymphopenia (0.53 × 10⁹ cells/L; RI: 0.7 to 5 × 10⁹ cells/L). Red cell parasites, such as Mycoplasma haemolamae, were not observed on microscopic evaluation of the blood smear. Band form eosinophils were present, an expected finding in camelids (12), and platelet numbers were normal. The most significant finding on CBC was the presence of a moderate number of single small, and multiple large, cytoplasmic inclusions within numerous neutrophils, consistent with Anaplasma phagocytophilum morulae.

Abnormalities on the chemistry profile included hyperproteinemia (73 g/L; RI: 60 to 72 g/L,) hyperalbuminemia (44 g/L; RI: 35 to 42 g/L) and increased beta-hydroxybutyrate (BHB) (105.6 μmol/L; RI: 28.8 to 67.2 μmol/L), moderate calculated hyperosmolality (315 mmol/kg; RI: 290 to 306.5 mmol/kg) and hyperglycemia (21.2 mmol/L; RI: 5.6 to 8.5 mmol/L.) Blood concentrations of lactate were increased (3.51 mmol/L; reference value < 2.5 mmol/L).

Based on hematological and serum chemistry analyses, therapy was aimed at treating presumed anaplasmosis, pyrexia, hyperglycemia, and dehydration, and providing nutritional support during sustained anorexia. Medications administered included oxytetracycline hydrochloride (Agrimycin 100; AgriLaboratories, St. Joseph, Missouri, USA),10 mg/kg BW, IV in 60 mL 0.9% sodium chloride q12h, thiamine hydrochloride (Vedco, St. Joseph, Missouri, USA), 10 mg/kg BW, SQ q12h, and flunixin meglumine (Prevail; VetOne, Meridian, Idaho, USA), 1.1 mg/kg BW, IV q12h for anti-inflammatory, analgesic, and anti-pyretic effects. As there are no pharmacologic data available for the dosing of oxytetracycline in New World camelids, the dose used was extrapolated from a previous report by Lascola et al (8), chosen due to its effectiveness against rickettsial diseases in camelids (8,9) and other species (1–3,8,9). One dose of short-acting regular insulin (Humulin N; Eli Lilly Indianapolis, Indiana, USA), 0.2 U/kg BW, SQ, was given along with a dose of long-acting Glargine insulin (Lantus; Sanofi-Aventis Bridgewater, New Jersey, USA), 0.4U/kg BW, SQ to up-regulate peripheral glucose uptake, stimulate hepatic glycogen and fatty acid synthesis, and inhibit intracellular lipolysis and ketogenesis (13–15). Intravenous fluids were administered [lactated Ringer’s solution (LRS) at 2 mL/kg BW per hour]. Partial parenteral nutrition (PPN) was administered in the form of 8.5% amino acids (Aminosyn; Hospira, Lake Forest, Ilinois, USA) added to the LRS, to counteract the alpaca’s negative energy balance. Blood glucose was monitored every 4 h initially and it gradually decreased to the normal range. Grass hay, alfalfa hay, and fresh grass were offered free choice, and the alpaca was hand-milked several times daily to stimulate milk production.

On day 2, the alpaca had an improved attitude, and body temperature and blood glucose were normal; however, she remained inappetent and passed no feces. Flunixin meglumine was decreased to 0.5 mg/kg IV BW, q12h and other therapies were continued. The animal began eating and drinking that night and passed normal formed feces. Blood glucose and vital parameters were normal until discharge. On day 3, milk production had resumed, appetite had improved, PPN was decreased to 1 mL/kg BW per day, and on day 5 the PPN and all medications were discontinued except oxytetracycline, which was given through day 7. A CBC was repeated on day 6 and showed a persistent mild anemia with an improved packed cell volume (0.25 L/L; RI: 0.27 to 0.4 L/L) and a lower hemoglobin concentration (92 g/L; RI: 105.5 to 173.4 g/L). No morulae were noted in the white cells on repeat CBC. Repeat chemistry profile showed no significant abnormalities. No abdominal distention was observed after abdominal drainage on day 1, and ventral and muzzle edema had resolved completely by day 7.

The alpaca was discharged with her cria on day 7, after the course of intravenous oxytetracycline was finished, and was sent home on long-acting oxytetracycline (Liquamycin; LA-200 Pfizer Animal Health New York, New York, USA), 20 mg/kg BW, SQ, q48h for 5 treatments. Recommendations were made to minimize tick exposure, and to have A. phagocytophilum titers performed on the rest of the herd, but this was declined. Polymerase chain reaction (PCR) testing (Vector Borne Diagnostic Laboratory, North Carolina State University, North Carolina, USA) on EDTA blood obtained on day 1 was positive for A. phagocytophilum. Paired serum samples were collected on day 1 and 5 wk later and submitted for serum IFA testing for A. phagocytophilum (Protatek International, Chandler, Arizona, USA). The acute phase titer on day 1 was negative and the convalescent titer 5 wk later was 1:1280.

The alpaca was reported to be doing well 4 mo after discharge; however, the same individual was presented to the University of Minnesota as a companion to a second premature cria 1 y after discharge. On this occasion the dam was bright, alert, and responsive with normal vital parameters and showed no abnormal clinical signs. Evaluation of the reproductive tract was declined by the owner. Repeat IFA testing for Anaplasma at this time revealed a negative titer and PCR testing for A. phagocytophilum, on ethylenediamine tetra-acetic acid (EDTA) blood, was negative as well.

Discussion

To the authors’ knowledge, this is the second report in the veterinary literature of A. phagocytophilum infection in an alpaca, and the first case in the midwestern United States. Support for the association of this animal’s clinical signs with Anaplasma infection include confirming the presence of the organism through light microscopy and PCR, demonstration of a serologic response to infection with A. phagocytophilum and the rapid resolution of clinical signs after treatment with oxytetracycline (2,3). The paucity of literature suggests that there may be a low prevalence of clinical infection in alpacas, or low detection of infection in camelid species. Our findings indicate that A. phagocytophilum may be associated with clinical signs of anemia, leukopenia, and fever in camelids in areas in which the organism is present.

The clinical signs associated with A. phagocytophilum infection in camelids in the 2 previously reported cases included lethargy, anorexia, decreased gastro-intestinal borborygmi, ataxia, recumbency, fever, and blindness (8,9). In the current case, infection with A. phagocytophilum was also associated with fever, lethargy, decreased borborygmi, and anorexia and, similar to previous reports, the animal responded favorably to treatment with oxytetracycline. However, the current case differed from the previous ones in that there was no evidence of ataxia or other neurologic deficits, and the clinical signs were a combination of facial and ventral edema, abdominal distention due to abdominal effusion, and premature parturition. Risk factors for premature parturition in alpacas are previous premature birth, abortion, twins, reproductive tract infection, stress, and being under or overweight at the time of parturition (16).

The pathogenesis of premature birth due to infection with A. phagocytophilum in other species is unclear. Abortion is not observed in pregnant mares (17); however, in small ruminant abortion due to infection with A. phagocytophilum, it has been speculated that the significant rise in body temperature, or the organism itself may be the cause (18). Alternatively, it has been suggested that A. phagocytophilum acts as an immunosuppressive agent that allows the development of secondary bacterial infections, resulting in abortion (7,19). The premature parturition in this alpaca may have been a manifestation of any of these mechanisms but as no examination of the reproductive tract was performed, other causes of premature parturition could not be ruled out. It is possible that the edema and peritoneal effusion seen in the alpaca in this report could have been due to endothelial inflammation associated with A. phagocytophilum infection (20,21). Unfortunately the placenta was not examined for evidence of vasculitis prior to presentation; however, edema is often seen in equids infected with A. phagocytophilum and vasculitis from Anaplasma infection could explain the edema in this case (1,3,17,22). No evidence of A. phagocytophilum morulae was found in a blood smear of the cria.

This alpaca had no risk factors for premature parturition on history or physical examination, other than stress from shearing. Typical hematologic abnormalities associated with A. phagocytophilum infection in the 2 previously reported cases and in other species include neutropenia, lymphopenia, anemia, and thrombocytopenia (3,8,9,22). In the current case the leukopenia and anemia may also have been associated with the A. phagocytophilum infection. In 1 of the previously reported cases (8), there was concurrent infection with Mycoplasma haemolama. Although M. haemolama was not observed in a blood smear in the current case, the presence of this organism was not tested for by PCR. In addition, moderate-to-severe anemia is frequently seen in sick camelids and often an underlying cause cannot be identified (12). The mechanism for the hematologic abnormalities observed in association with A. phagocytophilum infection is poorly understood, but in other species it has been suggested that after inoculation via tick-bite, the organisms invade hematopoetic and lymphoreticular cells after spread through the blood or lymphatic system (22). The organisms survive within neutrophils by altering host cell defenses through disruption of normal neutrophil function, such as inhibition of neutrophil superoxide production, decreases in neutrophil motility and phagocytosis, and the delay of neutrophil apoptosis (20). Impaired neutrophil function and leukopenia are thought to predispose to the development of opportunistic infections. Some research has demonstrated endothelial cell infection by A. phagocytophilum which could lead to endothelial inflammation and result in changes in vascular permeability (21). Immunologic studies with A. phagocytophila indicate both a cell-mediated and a humoral immune response to clinical infection (17); however, the immune response to infection is not well understood (21).

Following tick transmission of A. phagocytophilum in other species, morulae are found within neutrophils for only a short period of time in the acute phase of disease (17). In experimentally inoculated dogs, morulae appear 4 d after inoculation and have been noted to persist 4 to 8 d (2), and in horses they have been seen in 20% to 50% of neutrophils during days 3 to 5 post-infection (17). The PCR detection of A. phagocytophilum DNA is sensitive and specific for the diagnosis of the acute phase of anaplasmosis in other species (23), and confirmed the infection in this case. In the case reported here, sero-conversion in response to A. phagocytophilum antigens was documented by indirect immuno-fluorescent antibody testing. In other species affected by A. phagocytophilum a single acute phase serologic test may be negative, as sero-reactivity may not develop for 7 to 21 d (2,20). This was this alpaca’s first season in the midwest United States as she had been moved from a non-endemic region 4 mo prior to her premature delivery, and therefore may have been immunologically naïve to infection with this organism. Sero-prevalence of A. phagocytophilum in camelids is unknown in the midwestern United States, but individual strain and host variation may explain why clinical illness is observed in a small number of exposed animals. Little is known about long-term immunity after Anaplasma infection. Experimental studies in other species have shown that primary infection is followed by a variable degree of resistance (19,24). No information is available regarding persistence of infection in camelids; in this case infection was not detected at 1 y post-admission.

In conclusion, similar to other species, the clinical signs seen in association with A. phagocytophilum infection in camelids are variable and may include fever, edema, neurological signs, abdominal distention, ascites, lethargy, and anorexia (8,9). Parturient females in Anaplasma-endemic areas may be at increased risk of premature parturition or possibly abortion due to this disease. CVJ