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BRIEF COMMUNICATIONS and CASE REPORTS

Encephalitozoon cuniculi Associated Placentitis and Perinatal Death in anAlpaca ( Lama pacos )

J. D. W EBSTER , M. A. M ILLER , AND R. V EMULAPALLI

Animal Disease Diagnostic Laboratory and Department of Comparative Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayette, IN

Abstract. Placentitis, premature birth, and perinatal death were associated with Encephalitozooncuniculi infection in an alpaca. Histologically, chorionic trophoblasts contained many Gram-positive,period acidSchiff positive, variably acid-fast spores. Multifocal necrosis and infiltration bylymphocytes, eosinophils, and neutrophils were scattered throughout the chorionic membrane. Sporesin trophoblasts were approximately 1 mm 3 2 mm, thick-walled, and contained polar filaments and polarvacuoles consistent with microsporidia. The presence of E. cuniculi DNA was confirmed by sequencingthe polymerase chain reaction amplicon from frozen placental tissue. A few glial nodules were scatteredthroughout the cerebrum, and mild lymphocytic inflammation was present in the heart, liver, and lung.No organisms were detected in tissues other than the placenta. This is the first reported case of E.cuniculi infection in an alpaca.

Key words: Alpacas; camelids; Encephalitozoon cuniculi ; microsporidia; perinatal death; placentitis.

Encephalitozoon cuniculi is an obligate intracellularparasite of the phylum Microspora, which has beenassociated with spontaneous infections in numerousspecies. 17,20,21 In most species, E. cuniculi infections aresubclinical; however, clinical disease with nonsuppura-tive encephalitis, nephritis, vasculitis, pneumonia, hep-atitis, and placentitis can occur in neonatal, immuno-suppressed, and, less frequently, immunocompetentanimals. 21 To the authors knowledge, this is the firstdescription of placentitis, premature birth, and perinataldeath associated with E. cuniculi in an alpaca.

A reportedly 290-day-gestation female alpaca criaand placenta were submitted to the Animal DiseaseDiagnostic Laboratory at Purdue University for nec-ropsy on January 13, 2007. Reportedly, the cria wasdead when first seen the previous day. Its 3-year-oldprimiparous dam and other camelids on the farm wereclinically normal.

The cria weighed 3.8 kg and was 65.5 cm long fromcrown to rump. Based on the gestation length (normal 5335 to 360 days), 10 the weight (normal 5 approximately8 to 9 kg), 10 and unerupted teeth, the cria wasconsidered premature. The cria was in good bodycondition with moderate autolysis. The subcutis of thehead was edematous and mottled light to dark red. Thepleural and peritoneal cavities each contained approx-imately 200 ml of red, watery fluid. The lungs weremottled pink to dark red and oozed blood on cutsection. Sections of lung floated in formalin. Thechorionic surface of the placenta, especially near thebody, was mottled dark red to pink and covered withthick, 0.2-cm- to 0.4-cm-diameter, tangray, viscous,

loosely adherent plaques (Fig. 1). Near the umbilicalcord, the allantois was covered with similar exudate. Nogross lesions were observed in other tissues. Samples of placenta, lung, heart, brain, sciatic nerve, skeletalmuscle, spleen, thymus, lymph node, liver, stomachcompartments, small intestine, colon, kidney, andadrenal gland were fixed in 10% neutral bufferedformalin, routinely processed, paraffin embedded, andstained with hematoxylin and eosin for light microscopicexamination.

Many chorionic trophoblasts were distended with1 mm 3 2 mm, lightly basophilic, refractile spores withinparasitophorous vacuoles (Fig. 2). Necrotic foci werescattered throughout the edematous chorioallantois,which was infiltrated by scattered eosinophils, neutro-phils, and lymphocytes. The chorionic surface wassegmentally covered by amorphous eosinophilic materi-al, keratin, cellular debris, erythrocytes, neutrophils, andmany sloughed trophoblasts filled with spores. Thecerebral cortex had scattered glial nodules with a fewshrunken, hypereosinophilic neurons with pyknoticnuclei. In the lung, a few alveolar spaces containedsloughed epithelial cells, and there were scattered pleuralhemorrhages. Few lymphocytes, plasma cells, andneutrophils were scattered throughout pulmonary inter-alveolar septa, hepatic portal tracts, the endocardium,and the myocardium. Pyknotic and karyorrhecticlymphocytes were scattered throughout splenic periar-teriolar lymphoid sheaths.

Sections of placenta, cerebrum, and kidney werestained with Brown and Brenn modified Grams stain;placental sections were also stained with Ziehl-Neelson

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acid fast and periodic acidSchiff (PAS) stains. In theplacenta, spores were strongly Gram-positive, PAS-positive, and variably acid fastpositive, consistent withmicrosporidia and distinct from protozoa, such asToxoplasma gondii or Neospora caninum . No organismswere identified in other tissues.

One millimeter cubes of formalin-fixed placenta werefixed in 3% glutaraldehyde, postfixed in 1% osmiumtetrachloride, and embedded in Epon. Seventy- toninety-nanometer-thick sections were stained with leadcitrate and uranyl acetate, and examined with a Phillips201 electron microscope. The spores in trophoblasts hadsingle central nuclei, coiled polar tubes, thick walls, andposterior vacuoles, consistent with microsporidia(Fig. 3).

No bacteria or viruses were isolated from fetal tissues.Bovine viral diarrhea viral antigens were not detected byfluorescent antibody testing, and anti- Neospora anti-bodies were not detected by ELISA.

DNA from the frozen, unfixed placenta was extractedusing a commercial kit (DNeasy Tissue Kit, Qiagen,Valencia, CA) according to the manufacturers sug-gested procedure. Approximately 1 g of tissue washomogenized in 5 ml of deoxyribonuclease (DNase)-free water using a Stomacher, and a 160- ml aliquot of thehomogenate was used for DNA extraction. ExtractedDNA was eluted in 200 ml of kit AE buffer. Apolymerase chain reaction (PCR) with previously de-scribed primers 14 was performed on the extracted DNAto amplify the intergenic spacer region of genes encoding

Fig. 1. Placenta; alpaca. The chorion is mottled dark red to pink and covered with thick, loosely adherentplaques. Bar 5 2 cm.

Fig. 2. Placenta; alpaca. Chorionic trophoblasts contain many 1 mm 3 2 mm Gram-positive spores consistentwith E. cuniculi . Brown and Brenn modified Grams stain.

Fig. 3. Placenta; alpaca. A trophoblast contains a 1 mm 3 2 mm microsporidian spore with a coiled polarfilament (arrow). Lead citrate and uranyl acetate.

Fig. 4. Detection of E. cuniculi DNA in the alpaca on a 1% agarose gel stained with ethidium bromide afterPCR amplification. Numbers at the right indicate the 100-bp DNA ladder fragment sized in base pairs.

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small-subunit and large-subunit rRNA from severalmicrosporidian species. DNA extracted from cellculturegrown E. cuniculi spores (kindly provided byDr. R. Livingston, Research Animal Diagnostic Labo-ratory, University of Missouri, Columbia, MO) wasused as the positive control, and buffer was used asa negative control. The PCR was performed in a total

volume of 25 ml using 5 ml of the extracted DNAas template and primers MSP-1 (5 9-TGAATG(G/T)GTCCCTGT-3 9), MSP-2A (5 9-TCACTCGCCGCTACT-3 9), and MSP-2B (5 9-GTTCATTCGCACTACT-3 9). DNA amplification was performed ina thermocycler (Thermo Hybaid, Milford, MA) usingthe following protocol parameters: initial denaturing at95 u C for 5 minutes followed by 35 cycles of 94 u C for2 minutes, 55 u C for 2 minutes, 72 u C for 3 minutes, anda final extension at 72 u C for 5 minutes. Electrophoreticanalysis of 12 ml of the amplified products in 1.0%agarose gel revealed amplification of a , 400 bp DNAfragment from the placenta and the positive control; noamplified products were detected in the negative control(Fig. 4). The amplified DNA fragment from theplacenta was cloned into pCR2.1 vector (Invitrogen,Carlsbad, CA), and the nucleotide sequence of thecloned DNA fragment was determined at PurdueUniversity Low Throughput Genome Sequencing facil-ity. A basic local alignment search tool search of databases at the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov) revealedthat the amplified product was 98 to 100% identical tothe corresponding sequences of E. cuniculi . The presenceo f 4 5 9-GTTT-3 9 repeats in the amplified productindicated that E. cuniculi in the placenta belonged togenotype III. 7 Extracted DNA tested negative forToxoplasma gondii and Neospora caninum genomicsequences by real-time and nested PCR assays, re-spectively. 8,9

Infectious causes of camelid abortions have recentlybeen reviewed 19 ; however, few individual cases havebeen reported. Bovine viral diarrhea virus, Neosporacaninum , and Arcanobacterium pyogenes have beendirectly implicated in alpaca abortions. 3,12,18,19 Addi-tionally, maternal eosinophilic myositis, attributed toSarcocystis aucheniae infection, was associated withabortion with no fetal lesions. 15

Microsporidia are single cell, obligate intracellular,spore-forming parasites that are classified as fungi basedon the presence of chitin in spore walls and phylogeneticanalyses. 6 Microsporidia are characterized by thepresence of a polar filament, or polar tube, which isimportant for intracellular infection. 6,21

Microsporidia are important emerging human patho-gens, mainly infecting immunocompromised patients. 5,21Enterocytozoon bieneusi and Encephalitozoon intestinalisare most commonly associated with human disease 5,21 ;however, E. cuniculi has also been diagnosed in humanimmunodeficiency virus/acquired immunodeficiency dis-ease patients. 4 Encephalitozoon sp. have similarmorphologies, and PCR-based methods are necessaryfor species determinations. 5,21,22

E. cuniculi infections have been reported in rabbits,rats, mice, dogs, foxes, cats, squirrel monkeys, andhorses. 17,20,21 Microsporidia have direct life cycles andare transmitted mainly by ingestion and inhalation;however, traumatic and transplacental transmission alsohave been reported. 6,21 Transplacental E. cuniculi trans-mission has been proposed for several species, including

rabbits, carnivores, guinea pigs, horses, and squirrelmonkeys. 1,2,13,16,17,20,23

E. cuniculi associated placentitis has been reported insquirrel monkeys, 23 horses 17 (Armed Forces Institute of Pathology [AFIP] Wednesday Slide Conference [WSC]2004 # 23 [AFIP # 2890687] and AFIP WSC 1988 # 13[AFIP # 2133004]), and blue foxes. 16 In blue foxes 16 and1 equine infection (AFIP WSC 1988 # 13 [AFIP# 2133004]), the placenta contained organisms withfew other histologic changes, whereas 2 other infectedequine placentas 17 (AFIP WSC 2004 # 23 [AFIP# 2890687]) had multifocal necrosis with moderatenonsuppurative inflammation. Lesions were reportedlymore severe in squirrel monkeys with granulomatousplacentitis and vasculitis. 23 In this alpaca, placentallesions were moderate, with scattered necrotic foci andmoderate infiltration by lymphocytes, neutrophils, andeosinophils. Despite the difference in severity of lesions,these reports suggest that E. cuniculi can infect theplacenta of a variety of mammals and result in fetal loss.

Infected animals commonly shed E. cuniculi in urine,and the chitinous wall probably provides spores with theability to persist in the environment. 6 Recently, anti E.cuniculi antibodies were detected in 14.1% of Brazilianhorses, suggesting exposure to E. cuniculi .11 Because of its wide host spectrum and ability to survive in theenvironment, E. cuniculi should be considered asa potential cause of sporadic abortion and perinataldeath in multiple species, including camelids and horses.However, experimental studies fulfilling Kochs postu-lates are needed to confirm E. cuniculi s role in abortionand perinatal death.

Acknowledgements

We thank Phyllis Lockard for assistance with electronmicroscopy, and the Histopathology and MolecularDiagnostic Laboratories at Purdue University AnimalDisease Diagnostic Laboratory for histochemical stains,and PCR and sequencing, respectively.

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