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.

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.

Naturally acquired babesiosis in a reindeer (Rangifer tarandus tarandus) herd in Great Britain.

A provisional diagnosis of babesiosis was made in a reindeer herd in Scotland when seven animals died during 1997 and 1998. Additional clinical cases occurred, but the animals recovered after treatment. Thirty-one reindeer from the herd were tested for the prevalence of exposure to Babesia by the indirect fluorescent antibody test using a bovine isolate of Babesia divergens that had been passaged through gerbils. Infection rates were determined by Giemsa-stained blood smears. In addition, molecular identification of the infecting Babesiasp. was undertaken using SSU rRNA gene sequence analysis. It is likely that the organism causing babesiosis in this reindeer herd is B. divergens.

Genotypically unique Babesia spp. isolated from reindeer (Rangifer tarandus tarandus) in the United States.

Two morphologically dissimilar Babesia spp. were cultured from reindeer (Rangifer tarandus tarandus) in Placer County, Calif. The smaller isolate, designated RD61, was morphologically similar to Babesia odocoilei. Serum from RD61-infected reindeer reacted equally strongly to B. odocoilei and RD61 parasites in the indirect fluorescent antibody (IFA) test. Small subunit ribosomal RNA (SSU rRNA) gene-sequence analysis showed 99.0% identity to that of B. odocoilei. The larger piroplasm, designated RD63, resembled larger babesia organisms, such as Babesia caballi and Babesia bigemina. Serum from RD63-infected reindeer also reacted with both B. odocoilei and RD61 parasites in the indirect fluorescent antibody test. The SSU rRNA gene showed 94.2% identity to that of B. bigemina. Further studies are needed to determine whether these parasites are the same as the Babesia spp. previously documented in Siberian reindeer.

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.

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.

A study of the systematics of Theileria spp. based upon small-subunit ribosomal RNA gene sequences.

The systematics of benign and moderately pathogenic Theileria isolates from cattle and deer originating from different geographic regions was undertaken by small-subunit ribosomal RNA (SSU rRNA) gene nucleotide-sequence analysis. A maximum-likelihood phylogenetic tree constructed from these sequences resulted in two major divisions, each with a common ancestor. One major division branches into four relatively divergent groups, including (1) bovine Theileria sp. Type D (USA and Korea), (2) T. mutans Intona and Theileria sp. MSD (Africa), (3) T. cervi (USA), and (4) well-characterized pathogenic Theileria spp. (Africa). The other major division branches into two groups: (1) T. buffeli Warwick and T. buffeli Marula and (2) a second branch of closely related isolates with SSU rRNA gene Types B, B1, C, E, and H. Putative geographically associated diversity was noted only in the Korean bovine Theileria spp. with SSU rRNA gene types C and H and in African T. mutans Intona and Theileria sp. MSD. The current results show that the United States bovine Theileria isolates are not T. mutans because they have T. buffeli Marula (Type A) and/or Type D (species undesignated) SSU rRNA gene sequences. The taxonomic separation of T. buffeli Warwick from African T. mutans is confirmed in this study.

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.

Theileria sp. Infections associated with bovine fatalities in the United States confirmed by small-subunit rRNA gene analyses of blood and tick samples.

Theileria sp.-specific small subunit (SSU) rRNA gene amplification confirmed the presence of the organism in cattle and in Amblyomma americanum and Dermacentor variabilis ticks collected from a cattle herd in Missouri. Blood from the index animal had type A and type D Theileria SSU rRNA genes. The type D gene was also found in blood from two cohort cattle and tick tissues. The type A SSU rRNA gene was previously reported from bovine Theileria isolates from Texas and North Carolina; the type D gene was reported from a Texas cow with theileriosis.

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.

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.

Identical small subunit ribosomal RNA gene nucleotide sequence of bovine Theileria isolates (Korea and Japan) and Theileria buffeli (Marula, Kenya).

Small subunit ribosomal RNA (SSU rRNA) gene nucleotide sequences of bovine Theileria isolates from Korea (KLS and KCB) and Japan (JHS) were determined. The genes from each isolate were amplified by the polymerase chain reaction and the approximately 1.8 kb product cloned and sequenced by a modified dideoxynucleotide method. Overlapping gene segments produced with a series of primers were sequenced, resulting in a complete DNA sequence for both forward and reverse strands of the SSU rRNA genes of each isolate. SSU rRNA gene sequences (termed Type A) were identical among the bovine Theileria isolates from Korea and the isolate from Japan. A GenBank data library homology search showed the sequence to be the same as that listed as Theileria buffeli isolated from cattle in Marula, Kenya.

Case report: field-acquired subclinical Babesia equi infection confirmed by in vitro culture.

A horse with no prior clinical history of equine piroplasmosis tested negative for Babesia caballi and Babesia equi in the complement fixation test before importation into the United States from France. After 5 years in residence in the United States, the animal tested serologically positive for B. equi by the complement fixation test, the immunofluorescent antibody test, and Western blot analysis. The carrier status of the horse was confirmed by culture of B. equi parasites. In vitro culture offers an efficient and comparatively inexpensive method to determine the carrier status of horses suspected of harboring B. equi.

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.

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.

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.

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.

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.

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.

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.