Fasciola hepatica infections in cattle and the freshwater snail Galba truncatula from Dakhla Oasis, Egypt.

Infection by Fasciola species was investigated in seven districts of Dakhla Oasis, Egypt, through abattoir inspection of cattle livers for adult worms and sedimentation of faecal samples from local cattle to detect Fasciola eggs. In addition, lymnaeid snails collected from the study area were examined microscopically for developmental stages of Fasciola spp. Abattoir inspection revealed that 51 out of 458 cattle livers (11.1%) contained adult flukes, which were identified morphologically as Fasciola hepatica. Examination of the cattle faecal samples revealed that 142 out of 503 (28.2%) contained Fasciola eggs. The collected snails, identified as Galba truncatula and Radix natalensis, showed larval stages of Fasciola in 71 out of 731 (9.7%) G. truncatula, while R. natalensis showed no infection. Specific duplex polymerase chain reaction (PCR) targeting the mitochondrial cox1 gene of F. hepatica and Fasciola gigantica was carried out on DNA extracted from pooled infected snails and adult worms. The F. hepatica size amplicon (1031 bp) was obtained from both the infected G. truncatula and the adult worms isolated from cattle livers from different districts. The amplicon sequences were identical to the published sequences of F. hepatica mitochondrial cox1 gene. In conclusion, the zoonotic importance of Fasciola infection and appropriate hygienic measures must be taken into consideration in Dakhla Oasis, Egypt.

In vitro study of disinfectants on the embryonation and survival of Toxascaris leonina eggs.

The effect of six available and commercial disinfectants on the embryonation and larval development of Toxascaris leonina eggs was studied. Dettol® and Virkon® both induced a 100% reduction in larval development (P ≤ 0.05). Dettol® resulted in deformed eggshells and a halt in embryonal development at 1 week post exposure. All Virkon®-treated eggs showed an early embryonic lysis 24 h post exposure. TH4+ and 70% ethanol both significantly (P ≤ 0.05) affected larval development, with 58.8 and 85.8% reduction, respectively. Neither sodium hypochlorite nor phenol significantly affected larval development (2.8 and 21.0%, respectively). Sodium hypochlorite treatment caused a visible decortication of the eggshell; however, phenol-treated embryonated Toxascaris eggs appeared more or less morphologically normal. In conclusion, the disinfectants tested induced variable degrees of decortication and suppression of larval development. Virkon®S was the most effective disinfectant against Toxascaris eggs, suggesting that it is the most advisable one to use. To the best of our knowledge, this is the first report of the use of Virkon®S as an ovicide and/or larvicide of helminths, particularly Toxascaris leonina.

A common high molecular weight antigen of Babesia bovis isolates from Mexico.

Cattle from an area of Mexico endemic with Babesia bovis infections have a dominant antibody response to a 152kDa antigen of the Tamaulipas strain of B. bovis. A mAb termed PB/5, showing a specific reactivity to this 152kDa antigen in Western blots, was identified. The mAb which reacted with the blunt end of B. bovis in an indirect fluorescent antibody test also reacted to a 152kDa antigen in two other isolates (Nuevo Leon and Yucatan), and a 175kDa antigen in the Huasteca B. bovis isolate from Mexico. Polyclonal monospecific sera from a calf inoculated with mAb-affinity purified 152kDa antigen (Tamaulipas strain) identified B. bovis by the indirect fluorescent antibody test and two antigens of B. bovis (65kDa and 152kDa) in Western blot. Since the epitope reacting to the mAb PB/5 is conserved, this antigen provides a basis for developing a diagnostic test or an immunogen.

Derivation of monoclonal antibodies against Brucella abortus antigens.

In vivo immunization, fusion, antibody detection, and cryopreservation procedures for monoclonal antibody production against antigens of Brucella abortus are described. Splenocytes from BALB/c mice immunized with irradiated B. abortus S2308 were fused with Sp2/O-Ag14 myeloma cells and 61 hybridomas secreting anti-Brucella antibodies were cloned. Hybridoma antibody synthesis was detected effectively and most efficiently by enzyme-linked immunosorbent assays. Antibodies from clones of hybridoma A23 reacted with S19 and S2308 whole bacterial cells, while hybridoma B49 reacted primarily with alkali--treated lipopolysaccharides of S19, S1119.3 and S2308. Cryopreservation of clones had no major effect on antibody synthesis. The application of monoclonal anti-Brucella antibodies in the differential diagnosis of bovine brucellosis is discussed.

Biotin-labeled DNA probe in a PCR-based assay increases detection sensitivity for the equine hemoparasite Babesia caballi.

A DNA probe from Babesia caballi (Bc1) was selected by antibody screening of a genomic library. The Bc1 probe hybridized specifically to B. caballi genomic DNA. A polymerase-chain-reaction-based assay for B. caballi DNA was developed from primers deduced from the probe nucleotide sequence. An amplified product of 1.6 kb was detected from as little as 500 fg B. caballi template DNA. Sensitivity increased 1000-fold when the biotin-labeled Bc1 probe was hybridized to the amplicons in a Southern blot.

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.

In vitro cultivation of a Babesia isolated from a white-tailed deer (Odocoileus virginianus).

Pyriforms and ring forms of Babesia odocoilei were detected in thin blood smears obtained from a white-tailed deer killed by a hunter in Anderson County, Texas. Erythrocytes from the deer were cultured and the parasites maintained through 8 serial subcultures during 1 mo. The parasite was successfully established in culture using Medium 199 supplemented with either 20% deer serum or 40% normal adult bovine serum. The highest parasitemia observed was 30% and more than 4 parasites per erythrocyte were often observed. Cultured B. odocoilei remained infective for a susceptible white-tailed deer.

In vitro growth of Babesia bovis in white-tailed deer (Odocoileus virginianus) erythrocytes.

Babesia bovis cultured in bovine erythrocytes was passaged into white-tailed deer (Odocoileus virginianus) erythrocytes and medium containing either white-tailed deer serum or bovine serum. Deer erythrocytes supported the growth of the parasite only in the presence of bovine serum. Cryopreserved cultures were recovered successfully in white-tailed deer erythrocytes. By light and electron microscopy, B. bovis structure appeared similar in host cells of either species.

Isolation and in vitro cultivation of Babesia parasites from free-ranging desert bighorn sheep (Ovis canadensis nelsoni) and mule deer (Odocoileus hemionus) in California.

Protozoal parasites of the genus Babesia were isolated for the first time from free-ranging desert bighorn sheep (Ovis canadensis nelsoni) and mule deer (Odocoileus hemionus) populations in California by in vitro culture of host blood. These naturally infected animals did not have microscopically detectable parasitemia at the time blood was collected for parasite cultivation. Three isolates of small Babesia parasites were cultured from different sample groups of bighorn sheep, and 2 isolates of large Babesia parasites were cultured from a group of bighorn sheep and a group of mule deer, respectively. The size and structure of the various forms of piroplasms from each isolate remained consistent throughout the period of cultivation. Statistical comparison of the sizes of the piroplasms among the isolates indicated that there were at least 2 distinct morphotypes. Four of the 5 isolates were maintained with continuous growth in cultures containing erythrocytes from uninfected donor bighorn sheep, mule deer, and domestic sheep. Cryopreservation or storage of cultures at 4 C for 7 days did not affect viability of the isolates. These results demonstrate the potential for use of in vitro cultivation methods for the isolation of Babesia parasites from free-ranging artiodactylids.

Ultrastructural characteristics of Babesia odocoilei in vitro.

Babesia odocoilei continuously cultured in white-tailed deer (Odocoileus virginianus) erythrocytes was examined by transmission and scanning electron microscopy. Merozoites, trophozoites, intermediate-stage forms, and dividing forms were observed. Merozoites possessed a single nucleus, inner membrane complex, rhoptries, free ribosomes, rough endoplasmic reticulum, and single membrane-bound vesicles. Trophozoites lacked an inner membrane complex and rhoptries. Intermediate stages were characterized by distinct segments of inner membrane complex. Dividing forms ranged from cells with an elongated nucleus to mature daughter cells joined by a ringlike structure. Babesia odocoilei was characterized by its close proximity to the erythrocyte membrane, membranous structures resembling feeding organelles, and reproduction via a method resembling budding sensu stricto.

Babesia equi erythrocytic stage continuously cultured in an enriched medium.

Babesia equi was continuously cultured through 90 passages in an enriched chemically defined basal medium (HL-1) supplemented with 20% fetal bovine serum and serum replacement factors, including lipid-rich bovine serum albumin, bovine insulin, and human transferrin. Cryopreservation and subsequent recovery of B. equi were easily achieved. Inoculation of a splenectomized and an intact horse with cultured infected erythrocytes resulted in parasitemias and B. equi in vitro reisolation from both animals. In vitro forms of the parasite resembled in vivo forms. After establishment, parasitemias of 10-15% were commonly observed.

Babesia equi field isolates cultured from horse blood using a microcentrifuge method.

Babesia equi, a causative agent of equine piroplasmosis, was isolated from horses in the Chaco Province of Argentina, a known piroplasmosis endemic region. Fifteen B. equi field isolates were acquired by culture from 23 actively working horses from 2 ranches. The horses appeared healthy with no clinical signs or histories indicative of equine piroplasmosis. All 23 horses had B. equi-specific antibody activity by the indirect fluorescent antibody test and 18 were also complement fixation test positive for B. equi. Equine erythrocytes were prepared for parasite culture using a microcentrifuge tube method. This method greatly reduces the time involved in cell handling and parasite exposure to ambient conditions. By this method, B. equi cultures can be initiated from very small quantities of blood.

A new tick cell line derived from Boophilus microplus.

The Boophilus microplus IX tick cell line was developed from a primary embryonic cell culture derived from eggs six to nine days old. The cell line has been in culture since March 1979 and is currently maintained at 32 degrees C in medium consisting of equal parts of minimum essential medium an Leibovitz 15 medium supplemented with 20 per cent fetal bovine serum, 10 per cent tryptose phosphate broth and 0.1 per cent plasma albumin. A split ratio of 1:2 has been used for all subcultures and the cell line is now in its 26th passage. The cells are predominantly epithelial-like and of the male diploid chromosome number 21.

Ultrastructure of Babesia bovis sexual stages as observed in Boophilus microplus cell cultures.

Propagation of Babesia bovis in a Boophilus microplus cell line resulted in the appearance of the sexual stage of the parasite normally found only within tick intestine. These sexual stages, which possessed spike-like projections containing microtubules, were present in the medium and within cultured cells. Other ultrastructural characteristics of this sexual stage are described.

In vitro isolation and cultivation of a Babesia from an American woodland caribou (Rangifer tarandus caribou).

A Babesia species isolated from a captive caribou (Rangifer tarandus caribou) with clinical signs of babesiosis and a circulating parasitemia was cultured in vitro. Normal adult caribou erythrocytes supported the growth of the Babesia sp., as did erythrocytes from white-tailed deer (Odocoileus virginianus). Two basal media (M-199 and RPMI-1640) and a defined medium (HL-1) each supplemented with adult bovine serum were compared. The most favorable growth of the parasite occurred in HL-1 medium with 20% adult bovine serum. The morphology of this Babesia sp. isolate shared some characteristics with B. odocoilei and B. divergens.

Culture isolation and partial characterization of a Babesia sp. from a North American elk (Cervus elaphus).

Three North American yearling elk (Cervus elaphus) died with clinical symptoms suggestive of babesiosis. Babesia sp. organisms similar in morphology to B. odocoilei of white-tailed deer (Odocoileus virginianus) were observed in Giemsa-stained blood films from one of the elk. Continuous cultures of the parasite were established. Antiserum raised against the elk Babesia sp. isolate was compared to B. odocoilei specific antiserum in an immunofluorescent antibody assay; we found evidence of differences in reactivity to several Babesia spp. isolated from wildlife and domestic ruminants. Cultured parasites from the elk were not infective to either intact or splenectomized Bos taurus steers.

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.

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.

Culture confirmation of the carrier status of Babesia caballi-infected horses.

Culture of horse blood for Babesia caballi identified four carrier horses among nine previously infected horses. Three of the carriers had no detectable parasitemias on stained blood smears, and sera from two carrier horses were complement fixation test negative. Three cultures were continuously cultivated. Cryopreserved fourth-passage B. caballi was successfully reestablished in vitro. Blood from a 10th horse previously subinoculated with blood from a suspected carrier was cultured, with negative results.