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Veterinary Pathology Online http://vet.sagepub.com/ Theileriosis in a Missouri Beef Herd Caused by Theileria buffeli : Case Report, Herd Investigation, Ultrastructure, Phylogenetic Analysis, and Experimental Transmission S. L. Stockham, A. M. Kjemtrup, P. A. Conrad, D. A. Schmidt, M. A. Scott, T. W. Robinson, J. W. Tyler, G. C. Johnson, C. A. Carson and P. Cuddihee Vet Pathol 2000 37: 11 DOI: 10.1354/vp.37-1-11 The online version of this article can be found at: http://vet.sagepub.com/content/37/1/11 Published by: http://www.sagepublications.com On behalf of: American College of Veterinary Pathologists, European College of Veterinary Pathologists, & the Japanese College of Veterinary Pathologists. Additional services and information for Veterinary Pathology Online can be found at: Email Alerts: http://vet.sagepub.com/cgi/alerts Subscriptions: http://vet.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Downloaded from vet.sagepub.com by guest on February 6, 2011 Vet Pathol 37:11–21 (2000) ARTICLES Theileriosis in a Missouri Beef Herd Caused by Theileria buffeli: Case Report, Herd Investigation, Ultrastructure, Phylogenetic Analysis, and Experimental Transmission S. L. STOCKHAM, A. M. KJEMTRUP, P. A. CONRAD, D. A. SCHMIDT, M. A. SCOTT, T. W. ROBINSON, J. W. TYLER, G. C. JOHNSON, C. A. CARSON, AND P. CUDDIHEE Veterinary Medical Diagnostic Laboratory (SLS, DAS, MAS, GCJ), Department of Veterinary Medicine and Surgery (JWT), and Department of Veterinary Pathobiology (CAC), College of Veterinary Medicine, University of Missouri, Columbia, MO; Department of Pathology, Microbiology & Immunology, School of Veterinary Medicine, University of California, Davis, CA (AMK, PAC, TWR); and Heartland Animal Hospital, Ashland, MO (PC) Abstract. A 6-year-old Simmental cow infected with Theileria buffeli had a clinical disease characterized by theilerial parasitemia, macrocytic normochromic anemia with acanthocytosis and spherocytosis, lymphoid hyperplasia (lymphocytosis, edematous lymphadenomegaly), dysproteinemia, evidence of liver disease, and a low serum antibody titer against T. buffeli. The cow was in a herd in which all cattle originated in Missouri; 22/75 (29%) of cattle had a theilerial parasitemia and 26/75 (35%) had titers to T. buffeli of 1 : 160. Classi- ﬁcation of the Missouri bovine organism as T. buffeli was based on DNA sequencing and comparison to sequences for T. buffeli and Theileria sp. type A obtained from GenBank. Intraerythrocytic veils and piroplasms were seen during transmission electron microscopy. The organism was successfully transmitted to two splenec- tomized calves, which developed mild anemias while parasitemic. Blood from the second calf was used as the source of T. buffeli antigen for an indirect immunoﬂuorescence antibody test. Theilerial isolates from a Missouri white-tailed deer were also sequenced and resembled Theileria sp. types F and G and were not consistent with the bovine organism. Key words: Anemia; cattle; hemolysis; nuclear small subunit ribosomal RNA gene; phylogeny; protozoa; serology; Theileria; theileriosis; ultrastructure; white-tailed deer. Hemoparasites known to infect bovine erythrocytes The ﬁrst two reports of Theileria sp. seen in bovine and cause anemia include organisms from the genera erythrocytes in the United States originated in Kansas Anaplasma, Eperythrozoon, Babesia, and Theileria. (1950) and Texas (1975) and the organisms were iden- Theilerial parasites infect a broad range of wild and tiﬁed as T. mutans.18,29 In both reports, intraerythro- domestic artiodactyls throughout the world with high- cytic piroplasms were not considered pathogens and est prevalence in tropical and subtropical climates of were found in calves experimentally infected with An- Africa, Europe, Australia, and Asia.4,13 East Coast fe- aplasma marginale. More recently, cases of bovine ver, caused by T. parva, is the most severe theilerial theileriosis were reported in Texas and North Carolina. disease of domestic cattle. The life cycle of Theileria Infected cattle had parasitemias, but clinical signs were spp. includes intralymphocytic schizonts and intra- not reported.2 Based on small subunit ribosomal RNA erythrocytic piroplasms (merozoites and trophozoites). gene analysis of four isolates, the agents were consid- Intralymphocytic schizonts are the major pathogenic ered indistinguishable from or closely related to T. buf- stage for T. parva, erythrocytic piroplasms are the ma- feli. jor pathogenic stage for T. mutans, T. orientalis, and Here, we describe a severe case of bovine theileri- T. sergenti, and lymphocytic and erythroid forms of osis in Missouri, characterize the organism’s structural T. annulata are considered pathogenic.13,27 Piroplasms and molecular features, provide results of experimental of Theileria spp. are structurally very similar to those transfer, and report a high herd prevalence of theilerial of Cytauxzoon spp. and small Babesia spp.4,23 infection. This report is the most complete investiga- 11 Downloaded from vet.sagepub.com by guest on February 6, 2011 12 Stockham, Kjemtrup, Conrad, Schmidt, Scott, Robinson, Tyler, Johnson, Carson, and Cuddihee Vet Pathol 37:1, 2000 tion of bovine theileriosis in the United States to date. Blood was collected from 75 cattle for hematologic and Although bovine theileriosis had not been documented serologic evaluation. Blood ﬁlms were made immediately in Missouri, a theilerial agent is commonly seen in after collection of the blood samples in tubes with ethyl- Missouri white-tailed deer (Odocoileus virginianus).17 enediaminetetraacetic acid (EDTA). Later the same day, blood ﬁlms were stained with a Wright-Giemsa stain (Fisher Also, a closely related organism (similar to B. diver- Diagnostics). Two people (a medical technologist and a clin- gins, which infects cattle and people in Europe) is de- ical pathologist) examined a blood ﬁlm from each animal for scribed in Missouri as the cause of a lethal case of intraerythrocytic piroplasms for a minimum of 5 minutes for human babesiosis.9 each ﬁlm. If neither microscopist found intraerythrocytic piroplasms, the ﬁlm was considered negative. To be consid- Materials and Methods ered a positive ﬁlm, both microscopists had to ﬁnd at least Animals two parasitized erythrocytes. Index case. In mid-April, a referring veterinarian exam- Microhematocrits were measured by centrifugation of ined a 6-year-old pregnant Simmental cow that was lethargic EDTA-treated blood. Serum was collected from clot tubes and weak. She had separated herself from the rest of the and sent to the School of Veterinary Medicine, University herd for at least 2 days and recently weaned a 225-kg calf. of California–Davis, for determination of titers to the T. buf- The cow was in sternal recumbency but still eating, drinking, feli and to the National Veterinary Services Laboratory and chewing her cud (day 1). She could rise on her front (Ames, IA) for determination of titers to bovine leukemia legs but not on her rear legs. Other abnormalities included virus (agar gel immunodiffusion test) and A. marginale tachycardia and pale mucous membranes. Clinical signs (complement ﬁxation test). While blood was being collected, were suggestive of anaplasmosis, which is common in the veterinarians examined the cattle for evidence of lymphade- area. Blood was collected for hemoparasite detection and nomegaly, anemia, and the presence of ticks. The herd was serologic assays. A dextrose solution containing calcium was monitored by the MDA for additional cases of theileriosis administered intravenously, but no response was seen. Long- for 4–5 weeks. No additional clinical cases were seen, and acting tetracycline was given intramuscularly. Hay, water, the herd was released from quarantine. and grain were carried to the cow, and a portable corral was White-tailed deer. A 3-month-old white-tailed deer was installed around her. presented to the University of Missouri Veterinary Teaching The cow continued to eat and drink over the next few Hospital with diarrhea of 8 days duration, dehydration, and days. On day 6, the referring veterinarian examined the cow, a heavy tick infestation. The fawn was from a farm near the noted little change in the clinical status except more severe Missouri River, about 50 km downstream from the cow–calf weakness, and collected blood for analysis. index herd. Blood was collected for routine analysis and for On day 8, the cow was laterally recumbent, bloated, un- characterization of the intraerythrocytic piroplasms. Oxytet- able to stand, and moderately dehydrated. The rectal tem- racycline and ﬂuid therapy resulted in an uneventful recov- perature was 105 F, heart rate was 110 beats/minute, the re- ery. spiratory rate was 50 breaths/minutes, mucous membranes remained pale, and moderate scleral injection was present. Transmission electron microscopy Diarrhea was present, and the rectal wall felt thickened and Fresh whole blood from the index cow was processed for edematous. External lymph nodes, most notably the pre- transmission electron microscopy using methods previously scapular nodes, were enlarged. The cow appeared hyperes- described.31 thetic and her head and neck were pulled more dorsal than normal. Euthanasia was performed because of the cow’s de- Attempts to isolate Theileria sp. teriorating condition, and a necropsy was conducted. In vitro cultivation of the Theileria sp. was attempted with Splenectomized calves. Two male calves, 9 and 21 days whole blood samples from the index cow. Leukocytes were of age, were considered clinically healthy based on physical, removed from EDTA-anticoagulated blood from a healthy hematologic, and serum chemical data. Hemoparasites were cow by passage through a cellulose column. Erythrocytes not found during repeated blood analysis both prior to and were prepared as a 10% suspension in a solution containing after splenectomy. A splenectomy was completed on each 60% medium 199 with Earle’s salts and 40% fresh bovine calf by standard aseptic techniques.36 serum. The medium was buffered with TES [2-2(2-hydroxy- Herd of the index case. After the diagnosis of bovine 1-bis(hydroxymethyl amine)) ethanesulfonic acid], and pH theileriosis in one cow, the involved herd was quarantined was adjusted to 7.0. Blood from the index cow was added by the Missouri Department of Agriculture (MDA), and cat- to the normal erythrocytes in culture medium at a ratio of tle older than 4 months were examined by a consortium of one part test blood per four parts culture suspension. One- private, MDA, US Department of Agriculture, and Univer- milliliter aliquots were dispensed into wells of 24-well cul- sity of Missouri veterinarians. Other cattle in the cow–calf ture plates and incubated at 37 C in an atmosphere of 2% herd of about 45 cows and their offspring were not ill. The O2 and 5% CO2. Cultures were checked daily for 14 days herd was located within 1.5 km of the Missouri River ﬂood for parasite growth by examination of Giemsa-stained thin plain in central Missouri. All cattle in the herd were reared blood smears. by the owner except for two purebred bulls that were pur- Balb/c mice were inoculated intraperitoneally with blood chased within the two previous years; both bulls were raised from the index cow. Giemsa-stained ﬁlms of tail blood were in Missouri. examined daily for evidence of parasite growth. Mice were Downloaded from vet.sagepub.com by guest on February 6, 2011 Vet Pathol 37:1, 2000 Bovine Theileriosis 13 Table 1. Isolate name and GenBank accession numbers sequent hand alignment using MacClade 3.019 followed the of organisms compared by DNA analysis. secondary structure of the 18S small subunit ribosomal RNA (ss-rRNA) molecules for Theileria and Babesia obtained Isolate Name GenBank Accession No. from the ss-rRNA database.39 The ﬁnal alignment included Missouri cow AF162431 1,850 positions, all of which were included in the analysis. Missouri deer clone 1 AF162432 Cladistic analysis was performed using PAUP 3.1.34 Transi- Missouri deer clone 2 AF162433 tions and tranversions were weighted equally, and gaps were Theileria buffeli Z15106 treated as missing. Both a maximum parsimony approach Theileria sp. type A U97047 using a heuristic method with 100 bootstrap replications and Theileria sp. type B U97048 a branch-and-bound approach were used to generate phylo- Theileria sp. type D U97052 genetic trees. Theileria sp. type E U97053 DNA and phylogenetic analysis of Missouri’s cervine Theileria sp. type F U97054 theilerial agent Theileria sp. type G U97055 T. annulata M6243 Whole blood from the fawn was submitted to School of T. parva AF013418 Veterinary Medicine, University of California–Davis, for T. mutans (labeled clone 1) AF078815 parasite identiﬁcation. The 1.8-kb ss-rDNA from the white- Theileria sp. MSD strain AF078816 tailed deer was cloned because high background resulted (labeled clone 2) from initial sequencing. Amplicons for the 1.8-kb ss-rDNA Babesia divergens U16370 were ligated into a plasmid pCR 2.1 vector and transformed B. odocoilei U16369 into INV F One Shot Escherichia coli, according to the B. bovis L19078 manufacturer’s instructions (TA cloning kit, Invitrogen, San Neospora sp. U17345 Diego, CA). A kit (Quiaprep Spin Miniprep kit, Valencia, CA) was used to isolate cloned DNA. Presence of an insert was veriﬁed using an EcoR1 digest and visualization of the 1.8-kb product after agarose gel electrophoresis. Two clones monitored for overt clinical signs of illness and hematologic were chosen for sequencing. evidence of infection for 21 days. Experimental transmission to splenectomized calves DNA and phylogenetic analysis of T. buffeli Whole blood from the index cow was diluted with Vega Whole blood from the theileriosis cow was shipped to the y Murguia solution (VYMS), prepared as reported previous- School of Veterinary Medicine, University of California–Da- ly,41 and centrifuged at 1,000 g for 10 minutes. Superna- vis, for analysis. The Isoquick Nucleic Acid Extraction Kit tant was removed, and the pellet was combined at a 1 : 1 (Microprobe, Bothell, WA) was used as recommended by ratio with a 20% solution of polyvinylpyrolidone as cryo- the manufacturer to extract DNA from two 100- l aliquots protectant. One-milliliter aliquots of stabilate were frozen to of whole blood. The resultant DNA was pooled, and a 1.8- 70 C prior to storage in liquid nitrogen. kb nuclear small subunit ribosomal DNA (nss-rDNA) am- Aliquots were removed from liquid nitrogen, thawed at 37 pliﬁcation product was isolated using universal polymerase C, diluted in approximately 20 ml of 1 VYMS, and trans- chain reaction (PCR) primers.11 ferred to a syringe. Two aliquots were used for intravenous DNA cycle sequencing was performed on the 1.8-kb prod- injection and one aliquot was used for subcutaneous injec- uct using the ABI Prism 377 DNA sequencer protocol tion into the ﬁrst calf. Two aliquots were used for intrave- (Perkin-Elmer, Foster City, CA). The cow isolate was se- nous injection for the second calf. The inoculations occurred quenced directly from the PCR product. at 19 and 43 days of age and 4 and 19 days postsplenectomy The same universal primers were used to obtain nucleo- for calf Nos. 1 and 2, respectively. The calves were kept in tide sequence data from either end of the template. Subse- an indoor screened isolation facility after inoculation. The quently, internal primers were designed to allow for se- calves were monitored daily for clinical illness, and blood quencing the entire product in both the 5 to 3 and 3 to 5 samples were taken frequently to search for a theilerial par- directions (primer sequences available upon request). The asitemia and to measure hematocrits. Sera from postinocu- resulting chromatogram was read and analyzed using Se- lation days 7, 18, 42, 53, and 66 were shipped to the School quencer 3.0 (Genecodes Corporation, Ann Arbor, MI). of Veterinary Medicine, University of California–Davis, for The Missouri isolate was compared with 12 Theileria and determination of titers to T. buffeli. Babesia sequences (Table 1) obtained from GenBank be- cause of their genotypic similarity to the Missouri cow iso- Preparation of antigen slides for indirect immunoﬂuo- late or because they have been previously documented in rescence antibody (IFA) test for antibodies against T. animals in the United States.2 Two sequences of T. mutans buffeli obtained from cattle in Kenya and South Africa were also Whole blood was collected from the second experimen- included in the analysis. Neospora canimum, an apicom- tally infected calf on day 59 postinoculation and shipped plexan parasite also found in cattle in the United States,20 with ice packs overnight to California. At that time, 1–2% was used as an outgroup. of the calf’s erythrocytes contained intraerythrocytic piro- Sequences were initially aligned using Clustal W.35 Sub- plasms. Downloaded from vet.sagepub.com by guest on February 6, 2011 14 Stockham, Kjemtrup, Conrad, Schmidt, Scott, Robinson, Tyler, Johnson, Carson, and Cuddihee Vet Pathol 37:1, 2000 Fig. 1. Erythrocytes, lymphocyte, and platelets in peripheral blood; cow. Three erythrocytes (p) contain rod and racquet piroplasms. Spherocytes (s) and erythrocytes with basophilic stippling (b) are present. Wright’s-Giemsa stain. Same mag- niﬁcation as Fig. 2. Fig. 2. Erythrocytes and platelets in peripheral blood; cow. Spherocytes (s) and acanthocytes (a) are present. Wright’s- Giemsa stain. Bar 5 m. Fig. 3. Erythrocyte in peripheral blood; cow. Erythrocyte contains a cluster of theilerial cocci. Wright’s-Giemsa stain. Same magniﬁcation as Fig. 8. Fig. 4. Erythrocyte in peripheral blood; cow. Erythrocyte contains a theilerial rod and a Howell-Jolly body. Wright’s- Giemsa stain. Same magniﬁcation as Fig. 8. Fig. 5. Erythrocytes in peripheral blood; cow. Erythrocytes contain the common linear or comma-shaped piroplasms (upper cell) and many coccoid forms (lower cell). Wright’s-Giemsa stain. Same magniﬁcation as Fig. 8. Fig. 6. Erythrocytes in peripheral blood; cow. Erythrocytes contain larger, pleomorphic piroplasms. Wright’s-Giemsa stain. Same magniﬁcation as Fig. 8. Fig. 7. Erythrocytes in peripheral blood; cow. Erythrocytes contain a large signet-ring piroplasm (left) and small comma- shaped piroplasms (right); spherocyte also present (upper right). Wright’s-Giemsa stain. Same magniﬁcation as Fig. 8. Fig. 8. Erythrocytes in peripheral blood; cow. Three macrocytes are present; one contains small comma-shaped piro- plasms and basophilic stippling. Wright’s-Giemsa stain. Bar 5 m. The IFA antigen slides were prepared as previously de- contained Theileria piroplasms, including cocci, rod, scribed for B. gibsoni45 except that whole blood from ex- stick, comma, fusiform, racquet-shaped, signet-ring, perimentally infected calf No. 2 was used as the antigen and pear-shaped forms (Figs. 1, 3–8). Other erythro- source and rabbit anti-bovine ﬂuorescein isothiocyanate con- jugate (Jackson ImmunoResearch Laboratories, West Grove, cytic abnormalities included spherocytosis (Figs. 1, 2), PA) was used at a 1 : 300 dilution. Serum samples were di- acanthocytosis (Fig. 2), basophilic stippling (Figs. 1, luted in serial two-fold dilutions from 1 : 80 to 1 : 10,240. 8), Howell-Jolly bodies (Fig. 4), macrocytosis, and oc- casional schizocytes and keratocytes. There was a lym- Results phocytosis with reactive lymphocytes. Structural de- Animals fects were not detected in platelets. Index case. Initial blood ﬁlm examination revealed Complete blood cell count (CBC) results for a sam- anemia, thrombocytosis, and leukocytosis. Nearly 25% ple collected on day 6 included a macrocytic, normo- of erythrocytes on Wright-Giemsa-stained blood ﬁlms chromic, regenerative anemia, presence of many Thei- Downloaded from vet.sagepub.com by guest on February 6, 2011 Vet Pathol 37:1, 2000 Bovine Theileriosis 15 leria piroplasms, neutrophilia with mild left shift, lym- phocytosis, monocytosis, and hypoproteinemia (Table 2). Abnormalities in erythrocyte structure were still present. Schizonts were not found in blood lympho- cytes. Retrospectively, day 6 serum had a titer of 1 : 80 against the characterized T. buffeli using the IFA procedure developed during this study. In samples collected on day 8, intraerythrocytic piroplasms were present in 16% of erythrocytes, and CBC results revealed the persistence of anemia and an enhanced inﬂammatory response (Table 2). Abnor- malities in erythrocyte structure were still present, and the degree of spherocytosis was increased. Serum chemistry assays revealed a hypoproteinemia (4.3 g/ dl, reference interval [RI] 5.8–7.5 g/dl), hypoalbu- minemia (1.7 g/dl, RI 2.4–3.5 g/dl), hypocalcemia Fig. 9. Erythrocytes in peripheral blood; calf. No. 2. Eryth- (6.9 g/dl, RI 8.2–10.0 g/dl), hypomagnesemia (1.4 rocyte contains a large signet-ring and comma-shaped piro- mg/dl, RI 2.0–2.8 mg/dl), hyperbilirubinemia (0.8 plasms. Wright’s-Giemsa stain. Same magniﬁcation as Fig. 13. mg/dl, RI 0.1–0.6 mg/dl), increased serum aspartate Fig. 10. Erythrocytes in peripheral blood; white-tailed transaminase (AST), -glutamyl transferase (GGT), deer. Erythrocyte contains a cluster of four theilerial mero- and creatine kinase (CK) activities (729 IU/L, RI zoites in a quadruplet or Maltese-cross formation. Wright’s- 58–100 IU/L; 225 IU/L, RI 22–64 IU/L; 12,641 IU/ Giemsa stain. Same magniﬁcation as Fig. 13. L, RI 56–1,236 IU/L; respectively). Concentrations Fig. 11. Erythrocytes in peripheral blood; white-tailed of glucose, urea, creatinine, sodium, potassium, chlo- deer. Erythrocyte contains two large theilerial trophozoites. ride, globulins, and inorganic phosphates and total car- Wright’s-Giemsa stain. Same magniﬁcation as Fig. 13. Fig. 12. Erythrocytes in peripheral blood; white-tailed bon dioxide content were within reference intervals. deer. Erythrocytes contains large ring, small ring, and safety- Bovine leukemia virus antigen was detected (en- pin forms of theilerial piroplasms. Wright’s-Giemsa stain. zyme-linked immunosorbent assay) and complement Same magniﬁcation as Fig. 13. ﬁxation antibodies against B. bigemina and B. bovis Fig. 13. Erythrocytes in peripheral blood; white-tailed were not found in day 8 samples. Antibodies against deer. Erythrocytes contains several ring-forms of theilerial A. marginale were not detected by a latex agglutina- piroplasms. Wright’s-Giemsa stain. Bar 5 m. tion assay in a day 1 sample, but a weakly positive (1 : 5) complement ﬁxation test for A. marginale was ob- Table 2. CBC results from a Missouri cow with bovine theileriosis (samples from days 6 and 8 of clinical evaluation). Parameter Day 6 Day 8 Reference Intervals Plasma appearance Clear Clear Clear Plasma total protein (g/dl) 4.7* 5.4* 7.0–8.5 Fibrinogen (mg/dl) 500 900* 300–700 Packed cell volume (%) 13* 15* 24–46 Hemoglobin (g/dl) 4.45* 4.86* 8–15 Erythrocytes ( 106/ l) 2.0* 2.32* 5.0–10.0 Mean corpuscular volume (ﬂ) 64.9* 64.4* 40–60 Mean corpuscular hemoglobin concentration (g/dl) 34.3 32.5 26.0–34.0 Mean corpuscular hemoglobin (pg) 22.3* 20.9* 11.0–17.0 Leukocytes (cells/ l) 23,400* 27,300* 4,000–12,000 Neutrophils, segmented (cells/ l) 6,318* 6,006* 600–4,000 Neutrophils, band (cells/ l) 702* 2,457* 0–120 Lymphocytes (cells/ l) 14,976* 17,472* 2,500–7,500 Monocytes (cells/ l) 1,404* 1,365* 25–840 Eosinophils (cells/ l) 0 0 0–2,400 Basophils (cells/ l) 0 0 0–200 Platelets adequate clumped *Abnormal results. Downloaded from vet.sagepub.com by guest on February 6, 2011 16 Stockham, Kjemtrup, Conrad, Schmidt, Scott, Robinson, Tyler, Johnson, Carson, and Cuddihee Vet Pathol 37:1, 2000 tained for the day 8 sample. Marginal bodies of A. marginale were not found on any of the cow’s blood ﬁlms. Necropsy ﬁndings included a thin cow in early preg- nancy, edematous lymphadenopathy, splenic hemosid- erosis, acute pneumonia, subcutaneous edema, and thoracic and peritoneal effusions. An engorged Ambly- omma americanum (lone star tick) was found on the cow. Theilerial schizonts were not found in histologic sections or cytologic imprints of enlarged lymph nodes; however, postmortem autolysis interfered with analysis. There was no evidence of lymphosarcoma. A diagnosis of bovine theileriosis was based on ﬁnding many theilerial intraerythrocytic piroplasms, macrocytic anemia, and lymph node hyperplasia. Microscopic characteristics of bovine theilerial agent and infected erythrocytes The pleomorphism of the theilerial agent as seen via light microscopy was also evident via transmission electron microscopy. Infected erythrocytes contained piroplasms and veils (Figs. 14, 15). Attempts to culture Theileria sp. There was no in vitro propagation of organisms in bovine erythrocyte cultures as monitored by Giemsa- stained smears during a 14-day test period. Inoculated mice also showed no evidence of infection either by signs of clinical illness or hemoparasitemia. Fig. 14. Transmission electron microscopy. Theilerial infected erythrocyte, cow. Erythrocyte contains a theilerial Phylogenetic analysis of bovine agent piroplasm with acristate mitochondria (am), micronemes (m), nucleus (n), rhoptries (r), subplasmalemal membrane The relationship of the bovine isolate to other par- (sm), and veil (v). Uranyl acetate and lead citrate. Bar asitic protozoa is shown by the maximum parsimony 500 nm. approach with bootstrap support in Fig. 16. The branch-and-bound method (results not shown) pro- duced essentially the same relationship. tected in day 18 samples, and the serologic responses Theileria species, including the Missouri cow iso- increased during the study (Fig. 17). Calf Nos. 1 and late, form a monophyletic group separate from Babe- 2 were euthanized on days 66 and 67, respectively. sia. Thus, the Missouri cow isolate is not related to Calf No. 1 developed a mild respiratory infection the potentially zoonotic Babesia sp. found in a Mis- (viral or bacterial) soon after inoculation that pro- souri man in 1992. The cow isolate is nearly identical gressed slowly in severity; ceftiofur sodium was ad- to T. buffeli and Theileria sp. type A isolates. There ministered on days 25–30, after which there was is good statistical evidence to place T. annulata and marked clinical improvement. Marginal bodies of A. T. parva (the African theilerial species) in the same marginale were found on days 30 and 32. Eperyth- clade as the North American theilerial species. Two rozoon cocci were so numerous on days 32 and 35 clones of T. mutans are nearly identical to each other that they interfered with evaluation of erythrocytes for and are placed separately from the other theilerial spe- piroplasms. Marginal bodies and Eperythrozoon cocci cies. were not seen during the remainder of the evaluation. Calf No. 1 developed a mild anemia (day 39) concur- Experimental transmission to splenectomized calves rent with the ﬁrst detection of a theilerial parasitemia Theilerial piroplasms were found in the peripheral (Fig. 17). blood erythrocytes from days 39 to 66 postinoculation Shortly after the ﬁrst detection of theilerial parasit- in calf No. 1 and from days 35 to 67 postinoculation emia (day 35), calf No. 2 developed a slight anemia, in calf No. 2 (Fig. 9). Using the developed IFA test which progressed in severity (Fig. 17). Calf No. 2 re- for antibodies against T. buffeli, titers were ﬁrst de- mained clinically healthy during the study, and para- Downloaded from vet.sagepub.com by guest on February 6, 2011 Vet Pathol 37:1, 2000 Bovine Theileriosis 17 Fig. 15. Transmission electron micrograph. Theilerial in- Fig. 16. Dendogram showing evolutionary relationships fected erythrocyte, cow. Erythrocyte contains a theilerial of 18 Apicomplexan protozoal parasites. Branches are not piroplasm with acristate mitochondria (am), micronemes drawn in proportion to number of substitutions inferred to (m), nucleus (n), subplasmalemal membrane sm), and veil have taken place. Numbers above the branches represent (v). Uranyl acetate and lead citrate. Bar 200 mm. percentages of 100 maximum-parsimony bootstrap replica- tions in which the current groupings were obtaned. Bootstrap values 70% are considered good support for the grouping. In this analysis, values 70% are a result of minor pattern changes (branch swapping) in the terminal branches and do sites other than the theilerial piroplasms were not not affect the major conclusions. found except for rare marginal bodies of A. marginale in the day 35 sample. A peak theilerial parasitemia of 1–2% infected erythrocytes was found on day 56 post- inoculation; blood from day 59 was used to produce antigen slides for the T. buffeli IFA test. Herd investigation Twenty-two of 75 (29%) cattle had intraerythrocytic piroplasms but infected cells were rare ( 0.1% in- fected). One parasitemic cow had moderate lymphad- enomegaly; theilerial schizonts were not found in sev- eral smears of a lymph node aspirate. In 22 parasitemic cows, the mean packed cell volume (PCV) was 33% (range, 26–39%); in 16 cows in which intraerythro- cytic piroplasms were not found, the mean PCV was 32% (range, 27–39%). Dermacentor variabilis (Amer- ican dog tick) and A. americanum were found on sev- eral cattle while blood samples were being collected. Serologic titers against T. buffeli and prevalence of Fig. 17. Reciprocal titers (left axis) and hematocrit val- ues (right axis) in splenectomized calves inoculated with theilerial parasitemia are reported in Table 3. A kappa Theileria buffeli obtained from the index case. PCV, value24 of 0.78, implying a substantial agreement be- calf No. 1, piroplasms not detected; PCV, calf No. 1, yond chance between microscopic detection of para- piroplasms present; PCV, calf No. 2, piroplasms not sitemia and the IFA titer determinations, was derived detected; PCV, calf No. 2, piroplasms present; using 1 : 160 as a positive cutoff. Of the 22 parasitemic titer to T. buffeli in calf No. 1; h titer to T. buffeli in calf cattle, 19 had titers 1 : 160. Conversely, of 53 cattle No. 2. Downloaded from vet.sagepub.com by guest on February 6, 2011 18 Stockham, Kjemtrup, Conrad, Schmidt, Scott, Robinson, Tyler, Johnson, Carson, and Cuddihee Vet Pathol 37:1, 2000 Table 3. Assessment of herd prevalence of Theileria mia.22 Erythrocyte survival studies of calves infected buffeli infection by serologic testing and detecting theilerial with T. sergenti showed that both infected and unin- piroplasms during blood ﬁlm examinations (data do not in- fected erythrocytes have reduced survival.44 Studies clude index case). with T. annulata in calves suggested an immune-me- Theilerial Piroplasms diated hemolysis as indicated by the presence of a Titer to hemagglutinin.12 However, T. sergenti infections in se- Theileria buffeli Found Not Found verely combined immunodeﬁcient mice indicate that 1 : 80 3 46 the erythrocyte destruction can occur without immu- 1 : 80 0 3 noglobulin or complement involvement.8 The ﬁnding 1 : 160 1 3 that sera from cattle infected with T. sergenti and mer- 1 : 320 3 0 1 : 640 7 1 ozoite extracts both cause hemolysis led to the pro- 1 : 1,280 7 0 posal that proteases are involved in erythrocyte de- 1 : 2,560 1 0 struction.8 Proteases are involved in the invasion of erythrocytes by malarial and theilerial organisms.33 Oxygen radicals may also be involved in the patho- in which piroplasms were not detected, only four had genesis of the anemia.3 titers 1 : 160. Spherocytosis and acanthocytosis indicated that at Fifty-nine of 71 (83%) cattle had positive titers for least part of the cow’s anemia was due to erythrocyte bovine leukemia virus (BLV) and six of 46 (13%) had fragmentation. Acanthocytes, keratocytes, and spicu- a weak titer of A. marginale (four at 1 : 5, one at 1 : lated and spheroid cells have been reported in cattle 10, one at 1 : 20). Of six cattle with positive titers to infected with T. sergenti.10,43 Disseminated intravas- A. marginale, three had intraerythrocytic theilerial cular coagulation (DIC) can occur in theilerial and ba- piroplasms. Of 59 cattle that had positive BLV titers, besial infections,5,21 but it is unlikely that DIC was 20 had intraerythrocytic theilerial piroplasms. Of 12 present in this index case because thrombocytopenia, cattle that had negative BLV titers, two had intraeryth- hypoﬁbrinogenemia, and microthrombi were not rocytic theilerial piroplasms. found. Lymphoid hyperplasia was found in the index case Evaluation of white-tailed deer blood and DNA and is consistent with bovine theileriosis. Intralym- sequences phocytic theilerial parasites transform host cells and During the fawn’s illness, it had a PCV of 45% and lead to clonal growth of lymphocytes.26 The process had numerous intraerythrocytic piroplasms (Figs. 10– by which this transformation occurs is not known but 13). The theilerial agent was not considered a contrib- is associated with a growth factor produced by infected uting pathogen to the fawn’s illness. The deer isolates lymphocytes.7 Lymphocytic schizonts were not found were more closely related to Theileria sp. types F and in the index case; however, schizonts are considered G than they were to the cow isolate (Fig. 16). uncommon in cows infected with T. buffeli.30 Serum chemistry abnormalities seen in the index Discussion case are also consistent with bovine theileriosis. Hy- Theileriosis was considered the cause of the cow’s poproteinemia and hypoalbuminemia could be due to clinical illness, macrocytic normochromic anemia, and the extravascular accumlation of proteinaceous ﬂuids lymphoid hyperplasia. Macrocytosis, polychromasia, resulting from diseased lymph nodes22 and thus ex- and basophilic stippling indicated a regenerative re- plain the edema and body cavity effusions. Hypocal- sponse consistent with a hemolytic anemia. Splenic he- cemia and hypomagnesemia probably were due to the mosiderosis and the mild hyperbilirubinemia support- hypoproteinemia or decreased dietary intake. In- ed the hemolytic concept. A combination of sphero- creased serum GGT and AST activities, hypoprotein- cytosis, acanthocytosis, schizocytosis, and keratocy- emia, and mild hyperbilirubinemia could indicate liver tosis indicated intravascular erythrocyte fragmentation, disease, as seen with some theilerial infections,42 but as can occur with vasculitis or intravascular thrombi. postmortem liver lesions were not found. At least part Spherocytes can also be formed by immune-mediated of the hyperbilirubinemia was caused by hemolysis or processes. Undoubtedly, the inﬂammatory disease also anorexia. Increased serum AST and CK activities re- contributed to the anemia. There was no evidence of ﬂected muscle trauma caused by prolonged recumben- blood loss, but there was a hypoproteinemia. cy. The pathogenesis of anemia is not clearly estab- Prior to molecular characterization, taxonomic clas- lished in theileriosis and may be multifaceted. Invasion siﬁcation of Theileria spp. was based on microscopic of erythroid precursors by the merozoites of T. parva appearance of intraerythrocytic piroplasms, geographic is associated with erythroid hypoplasia and severe ane- location of the infected animal, apparent pathogenicity Downloaded from vet.sagepub.com by guest on February 6, 2011 Vet Pathol 37:1, 2000 Bovine Theileriosis 19 of the organism, and serologic testing.13,37 Most thei- by an MDA veterinarian did not reveal suspect cases. lerial organisms of cattle that had low pathogenicity However, the disease may be difﬁcult to differentiate were called T. mutans.30 As more Theileria spp. were clinically from anaplasmosis without blood ﬁlm ex- isolated and studied, some organisms were reclassiﬁed. aminations. The reason one cow developed a clinical For example, the US theilerial organism reported by illness while the other infected cattle in the herd re- Kuttler in 1975 to be T. mutans was renamed T. ori- mained clinically healthy is unknown. A combination entalis in 1985.38 These authors also considered some of predisposing factors could have inﬂuenced the isolates of T. orientalis to be the same as some stocks course of this disease; prednisolone and pregnancy of T. sergenti, the name given to a more pathogenic have been associated with enhanced T. sergenti para- species found in southeastern Asia. sitemia and associated illness.14,28 The fact that the cow In the index case, intraerythrocytic piroplasms were had a relatively low titer (1 : 80) against T. buffeli sug- highly pleomorphic, but rod forms were most com- gests either recent infection or a poor immunologic mon. Piroplasm shape may vary with stage of an in- response. fection and thus is not a reliable criterion for species Experimentally infected splenectomized calves did differentiation.37,46 Veils were not seen on Wright-Gi- develop mild to moderate anemia. Interpretation of emsa-stained blood ﬁlms, but veils were seen during data from calf No. 1 is difﬁcult because of the respi- electron microscopy. Electron-dense veils have been ratory disease that was present during the evaluation reported in erythrocytes infected with T. velifera, T. period and the appearance of Eperythrozoon sp. and sergenti, T. buffeli, and T. orientalis.32 The veil con- A. marginale shortly before detection of the theilerial tains hemoglobin40 and other proteins that appear to be piroplasms. Calf No. 2 remained clinically healthy, and of parasite origin.32 A. marginale was seen only on 1 day during the study, The Missouri cow and deer isolates are clearly part suggesting its anemia may have been due to theileri- of the Theileria spp. complex that has been docu- osis. It was assumed that the inoculate contained all mented worldwide. The extreme genetic similarity of three hemoparasites, but the calves could have been the cow isolate’s 18S ss-rRNA gene to that of T. buffeli infected with the nontheilerial parasites previously. (99.88% or 2-bp differences) as well as its clinical and In summary, a cow infected with T. buffeli had a morphologic manifestations strongly support the char- clinical disease characterized by theilerial parasitemia, macrocytic normochromic anemia with spherocytosis, acterization of this isolate as T. buffeli. This and other acanthocytosis, and other erythrocyte abnormalities, studies2 have demonstrated that T. buffeli is a clinical lymphoid hyperplasia, dysproteinemia, evidence of entity in North American cattle. liver disease, and a low titer against T. buffeli. The The role of wildlife as a reservoir for T. buffeli is anemia’s pathogenesis was not determined, but evi- not clear from this analysis. Our study showed that the dence suggested acanthocytic fragmentation hemoly- isolates obtained from the Missouri white-tailed deer sis. The cow was in a herd in which all cattle had been are more closely related to isolates previously found raised in Missouri, and the T. buffeli was considered in deer and elk in the United States and Canada than endemic based on serologic and microscopic blood ex- to T. buffeli. Therefore, deer do not appear to be the amination results. source of infection for this herd. DNA sequences were not available for T. cervi, a commonly reported thei- Acknowledgements lerial species in deer from the southern and southeast- We acknowledge the cooperation and patience of the ern areas of the United States.6,15 Thus, we cannot herd’s owner during our investigations. We also thank Dr. comment on the relationship of T. cervi to this bovine John Hunt and his staff, MDA, and Dr. Glen Snider, US Theileria isolate. The North American cervid organism Department of Agriculture, for their cooperation and dedi- cation during the investigation of the infected herd and for was classiﬁed as T. cervi in 1962,25 but failure of trans- providing serologic testing for A. marginale and BLA. We mission to fallow deer16 indicated that the organism is thank Howard Wilson for assistance and guidance in creating not the same as the T. cervi originally reported in Eu- photographic images, Preston Stogsdill for electron micros- rope.1 Bovine and cervid isolates from Missouri ani- copy assistance, Erdal Erol and Craig Bailey for attempts to mals were grouped within the Theileria clade and thus culture, isolate, and cryopreserve the theilerial agent, and the are not related to the zoonotic Babesia divergens-type technical staff of the Clinical Pathology Laboratory for the piroplasm found in a human patient from Missouri.9 analysis of blood samples. The work was supported in part T. buffeli was endemic in the cattle herd, as docu- by a grant from the Committee on Research, College of Vet- mented by the prevalence of parasitemia (29%) and erinary Medicine, University of Missouri–Columbia. the detection of T. buffeli antibodies (35%). 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