Theileriosis in a Missouri Beef Herd Caused by Theileria buffeli by bestt571

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    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

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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

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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


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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.


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   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-


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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.



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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-


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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.


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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


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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%). The prev-                                                   References
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Request reprints from Dr. Steve Stockham, A336 Clydesdale Hall, College of Veterinary Medicine, Columbia, MO 65211
(USA). E-mail: stockhams@missouri.edu.


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