G.G. Wagner

NUCLEOTIDE SEQUENCE HETEROGENEITY IN THE SMALL SUBUNIT RIBOSOMAL RNA GENE VARIABLE (V4) REGION AMONG AND WITHIN GEOGRAPHIC ISOLATES OF THEILERIA FROM CATTLE, ELK AND WHITE-TAILED DEER

The phylogenetic relationships among fourteen isolates of benign Theileria spp. infecting cattle, elk and white-tailed deer were studied by nucleotide sequence comparisons of the variable (V4) region (200 nucleotides) of the small subunit ribosomal RNA gene. Included were six Korean bovine, one Japanese bovine, three North American bovine, and four North American cervine isolates. The SSU rRNA gene from each isolate was amplified, cloned, and the V4 region fragment sequenced. Seven different nucleotide sequence patterns were obtained and classified. Type A was identical to T. buffeli SSU rRNA gene sequence (GenBank Accession No. Z15106) and was found in Korean, Japanese, and North American bovine isolates. Type B was found in bovine isolates from Korea, Japan and North America. Type C was found only in the Korean bovine isolate from Chungnam. Type D was found in a Korean and in a North American bovine isolate. Type E was found in a bovine isolate from Cheju Island of Korea and a North American cervine (elk) isolate. Types F and G were found only in North American cervine isolates (both white-tailed deer and elk) and appear to represent a species separate from the bovine isolates. The presence of several sequence types observed in most of the bovine Theileria isolates may indicate mixed species (or subspecies) populations and/or multiple genotypes within a single species.

Studies on the effect of infection by Babesia sp. on oviposition of Boophilus microplus engorged females naturally infected in the Mexican tropics.

Three hundred and fifteen engorged female Boophilus microplus ticks (weight 210-250 mg) naturally infected with Babesia sp., in the Mexican tropics were monitored for egg production. Haemolymph samples were taken from each tick on the 5th day to the 16th day after collection to detect and estimate the infection with Babesia sp. kinetes. All ticks were held in darkness at 27+/-1.5 degrees C and 85-86% relative humidity. The infection rate of Babesia sp. was 20.3% (64/315). Fifteen ticks were considered heavily infected and 49 lightly infected. The pre-oviposition periods were 3.17+/-0.37, 3.18+/-0.25 and 3.17+/-0.25 days for heavily infected, lightly infected and uninfected, respectively (P>0.05). The numbers of eggs laid on the first day of oviposition were 252+/-53, 235+/-37, 54+/-23 for heavily infected, lightly infected and uninfected, respectively. There was a statistically significant difference between infected (heavily and lightly) and uninfected (P<0.05) ticks. Oviposition periods were 9.60+/-0.81, 9.50+/-0.72 and 9.36+/-0.48 days for heavily infected, lightly infected and uninfected, respectively (P>0.05). The average egg production of heavily infected, lightly infected and uninfected female ticks was 2640+/-103, 2574+/-123 and 2841+/-170 (P>0.05), respectively. These data imply that there is an adaptative tolerance between Babesia sp., and B. microplus under field conditions in the Mexican tropics.

Antigenic, phenotypic and molecular characterization confirms Babesia odocoilei isolated from three cervids.

Babesia isolates from an elk (Cervus elaphus canadensis) and a caribou (Rangifer tarandus caribou) with fatal infections were compared to Babesia odocoilei (Engeling isolate) from white-tailed deer (Odocoileus virginianus) by experimental infection, serologic, and small subunit ribosomal RNA (SSU rRNA) gene sequence analysis studies. Both the indirect fluorescent antibody test and immunoprecipitation assays demonstrated antigenic variation among the isolates. Experimental infection studies showed no clinical differences among the isolates. Nucleotide sequence analysis showed that the elk and caribou Babesia sp. isolates possessed SSU rRNA genes with identical sequences to that of B. odocoilei. A phylogenetic tree constructed from SSU rRNA gene sequences shows that B. odocoilei is most closely related to Babesia divergens, both of which branch together in the true babesia clade.

The effect of management factors on the seroprevalence of Anaplasma marginale in Bos indicus cattle in the Mexican tropics.

A cross-sectional study with a two-stage design and proportional distribution was carried out to determine the effect of management factors on the seroprevalence of Anaplasma marginale in Bos indicus cattle in the Mexican tropics. Serum was obtained from 384 cattle aged 1– 2 years on 92 farms. The number of samples was proportionally distributed according to the number of farms in eastern Yucatan. Antibody activity against A. marginale was assessed using the card agglutination test. A primary screening using a 2×K contingency table of the exposed variables with the outcomes was performed. All variables for which p<0.20 were included in a fixed-effects log regression. The seroprevalence in the cattle was 69.75% (SE±0.02). Sixty-four per cent of the farms had a seroprevalence ≥75%. The risks related to managemental factors were stocking density (≥1 animal/ha, OR = 10.94), type of acaricide (pyrethroids, OR = 3.8), dipping interval (>60 days, OR = 0.13) and type of veterinary instruments used (needles, scalpels, ear tattoos, and dehorners, OR = 0.17).

TWO THEILERIA CERVI SSU RRNA GENE SEQUENCE TYPES FOUND IN ISOLATES FROM WHITE-TAILED DEER AND ELK IN NORTH AMERICA

Two Theileria cervi SSU rRNA gene sequence Types, F and G, from white-tailed deer (Odocoileus virginianus) and elk (Cervus elaphus canadensis) isolates in North America were confirmed. Previously, nucleotide sequencing through a single variable (V4) region showed the presence of SSU rRNA gene Types F and G in T. cervi isolates from white-tailed deer and an elk. In this study, both sequence types were found in four T. cervi isolates (two from deer and two from elk). Microheterogeneity only appeared in the Type G gene, resulting in Subtypes G1, G2 and G3. Subtype G1 was found in two elk and one white-tailed deer T. cervi isolate; Subtypes G2 and G3 were found in a white-tailed deer T. cervi isolate. The Type F SSU rRNA genes were identical in nucleotide sequence in both elk and white-tailed deer T. cervi isolates. The high degree of conservation in the Type F variable regions may be exploited to design specific oligonucleotide primers for parasite detection by the polymerase chain reaction in cervine or tick hosts.

The Isolation and Partial Characterization of A Babesia Sp. From Desert Bighorn Sheep (Ovis Canadensis Nelsoni)

ABSTRACT. A novel Babesia parasite of desert bighorn sheep was isolated. Its taxonomic description, host range, pathogenicity and antigenic relatedness were in vestigated. the parasite was infective for black‐tailed and white‐tailed deer, but with host‐specific differences compared to that of bighorn sheep. A splenectomized calf and domestic sheep were refractory to infection. A comparative immunofluorescence assay detected antigens cross‐reactive with Babesia odocoilei, B. divergens, B. equi and B. caballi, but not with B. bovis or, B. bigemina. Babesia odocoilei was also infective for bighorn sheep, allowing comparison by a cross‐challenge experiment, the results of which supported the conclusion that this parasite was not B. odocoilei. However, the bighorn sheep Babesia cannot currently be distinguished from B. capreoli described from roe deer in northern Germany. Data indicate that, while this parasite may not present a problem for domestic animals, it may cause disease in bighorn sheep and deer populations.

Transmission of Babesia odocoilei in white-tailed deer (Odocoileus virginianus) by Ixodes scapularis (Acari: Ixodidae)

Laboratory reared Ixodes scapularis proved to be an efficient vector of Babesia odocoilei Emerson and Wright between white-tailed deer (Odocoileus virginianus). Transta-dial survival of the babesia occurred between nymph and adult stages of the tick, and the adult stage transmitted the babesia.

Increased activity of bovine ADCC effector cells during acute Babesia bovis infection

Mononuclear effector cells from the peripheral blood of Babesia bovis-infected cattle responded during initial infection and destroyed Fc-bearing target cells. The activity of these cells was measured in an antibody-dependent, cell-mediated cytotoxicity (ADCC) assay utilizing chicken erythrocytes coated with bovine anti-chicken erythrocyte antibody as targets. The activity of these effector cells was enhanced during the parasitemic crisis and returned to normal levels of activity following the resolution of parasitemia. This suggests that ADCC mechanisms may be involved in resolution of infection of cattle with B. bovis.

Development and validation of an indirect enzyme immunoassay for detection of antibody to Anaplasma marginale in bovine sera

An indirect enzyme immunoassay (IELISA) for detection of bovine antibody activity to Anaplasma marginale was developed. This assay used a crude antigen prepared from erythrocytes of infected calves, immobilized in a polystyrene matrix and a mouse monoclonal antibody to bovine IgG1, conjugated with horseradish peroxidase. Negative sera (n = 1842) were tested and the diagnostic specificity was 98.4 +/- 0.6% before retesting 29 positive samples. After retesting, eight samples remained positive and the specificity was calculated to be 99.6 +/- 0.3%. The diagnostic sensitivity, using 831 serum samples collected from naturally or experimentally infected cattle in Argentina, 370 from Mexico and 525 sera from experimentally vaccinated or infected cattle from Texas, was 87.3 +/- 1.6%.

Serological prevalence and isolation of Babesia odocoilei among white-tailed deer (Odocoileus virginianus) in Texas and Oklahoma.

Serum samples collected from 581 white-tailed deer (Odocoileus virginianus) from Texas and from 124 white-tailed deer from Oklahoma were tested by the indirect fluorescent antibody technique against Babesia odocoilei. Prevalence of seropositive reactors varied from site to site in both states. Prevalence rates were statistically ranked as high, intermediate or low. Deer <12-mo-old had a significantly lower prevalence than all other age classes.