Seroprevalence estimation and management factors associated with high herd seropositivity for Babesia bovis in commercial dairy farms of Puerto Rico.

A cross-sectional study was conducted to determine individual cow seroprevalence of Babesia bovis in adult lactating dairy cattle of Puerto Rico (PR), to assess the associations of farm management factors on herd seroprevalence, and to document the species of ticks infesting cattle within these farms. Antibody activity against B. bovis was determined using an indirect fluorescent antibody test (IFAT). Serum samples were obtained from 2,414 adult lactating dairy cattle from 76 randomly selected commercial dairy farms. Herd seroprevalence ranged from 0 to 51% with an overall individual cow seroprevalence for B. bovis of 26%. Ticks were collected from animals on 7 (9%) of the 76 participating commercial dairy farms. All collected ticks (n = 87) were Rhipicephalus (Boophilus) microplus. Factors associated with high herd seropositivity were dairy farms with calf but not heifer raising facilities (OR = 16, 95% CI = 3.0-86), having more than 4 neighbors with cattle (OR = 17, 95% CI = 1.6-178), same producer owning more than one farm (OR = 7.2, 95% CI = 1.6-32), and use of government services to apply amitraz on cattle (OR = 5.5, 95% CI = 1.5-20).

A single-chain fragment variable recombinant antibody against F5 fimbria of enterotoxigenic Escherichia coli inhibits agglutination of horse red blood cells induced by F5 protein.

Bovine colibacillosis caused by enterotoxigenic Escherichia coli (ETEC) is a worldwide problem. Adhesion of ETEC to intestinal cell receptors mediated by the surface protein F5 fimbriae is the initial step in the establishment of colibacillosis. Prevention of ETEC F5(+) adhesion to enterocytes protects newborn calves against collibacillosis. On the enterocytes, the F5 fimbriae bind to a ganglioside that is also found on horse red blood cells. Thus, the presence of F5 fimbriae induces haemagglutination, which is useful as an indicator in a functional assay system. In this study, recombinant anti-F5 scFv antibody fragment produced in E. coli HB2151 reacted with F5 fimbriae in ELISA and Western immunoblot, and prevented haemagglutination induced by the binding of the F5 fimbriae to its natural host receptors on horse red blood cells. Given the ease with which recombinant antibodies can be mass-produced, the presently described scFv may hold promise as a prophylactic agent for colibacillosis.

Antibodies: an alternative for antibiotics?

In 1967, the success of vaccination programs, combined with the seemingly unstoppable triumph of antibiotics, prompted the US Surgeon General to declare that "it was time to close the books on infectious diseases." We now know that the prediction was overly optimistic and that the fight against infectious diseases is here to stay. During the last 20 yr, infectious diseases have indeed made a staggering comeback for a variety of reasons, including resistance against existing antibiotics. As a consequence, several alternatives to antibiotics are currently being considered or reconsidered. Passive immunization (i.e., the administration of more or less pathogen-specific antibodies to the patient) prior to or after exposure to the disease-causing agent is one of those alternative strategies that was almost entirely abandoned with the introduction of chemical antibiotics but that is now gaining interest again. This review will discuss the early successes and limitations of passive immunization, formerly referred to as "serum therapy," the current use of antibody administration for prophylaxis or treatment of infectious diseases in agriculture, and, finally, recent developments in the field of antibody engineering and "molecular farming" of antibodies in various expression systems. Especially the potential of producing therapeutic antibodies in crops that are routine dietary components of farm animals, such as corn and soy beans, seems to hold promise for future application in the fight against infectious diseases.

Histochemical localization of esterases in the integument of the female Boophilus microplus (Acari: Ixodidae) tick.

The cattle tick Boophilus microplus (Canestrini) is one of the most important ectoparasites affecting tropical cattle with worldwide distribution. Application of organophosphate compounds (OP) is extensively used as a tick control method. However, the appearance of ticks resistant to the OP decreases the therapeutic efficacy of such compounds. Esterases have been implicated as potential biochemical mechanisms for detoxification in B. microplus larvae. We found increased esterase activity in the inner layers of the integument of OP resistant adult female B. microplus ticks as compared with the OP susceptible ticks. We discuss the potential role of these enzymes during acaricide metabolism and propose future research.

Antigenic analysis of Anaplasma marginale grown in bovine erythrocytes co-cultured with bovine endothelial cells.

Two monoclonal antibodies (mAbs) for A. marginale were used to test the antigenic integrity of A. marginale grown in vitro in bovine erythrocytes co-cultured with endothelial cells. Both the mAbs reacted in the indirect immunofluorescent antibody test with A. marginale grown in vitro and also detected the antigens in Western immunoblots of SDS-PAGE separated antigens made from A. marginale infected erythrocytes from the cultures. Furthermore, active replication was evident as [35S]-methionine is incorporated by A. marginale present in the second passage of a culture maintained for six weeks as shown by immunoprecipitation of labeled antigens by the mAbs. This indicates that A. marginale grown in the in vitro culture system described previously [Waghela et al., Vet. Parasitol. 73 (1997) 43] maintain antigenic character, and with further development the system can be used for preparing immunogens or diagnostic antigens.

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.

Identification of a point mutation in an esterase gene in different populations of the southern cattle tick, Boophilus microplus.

Two esterase cDNA sequences were obtained from susceptible and organophosphorus resistant strains of Boophilus microplus. Both sequences have a high degree of homology to carboxylesterase B. One gene has identical sequences in both strains and the other showed two point mutations. One mutation produces an amino acid substitution when the amino acid sequence is deduced, this mutation was detected in six different populations susceptible and resistant to insecticides, but a pyrethroid resistant strain was the only one that showed only the mutant allele. Identification of this mutation and the strong signal detected in southern blot with this strain, suggest that esterases are contributing to detoxification of pyrethroid compounds, as a resistant mechanism in Mexican strains of the southern cattle tick.

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.

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.

In vitro cultivation of Anaplasma marginale in bovine erythrocytes co-cultured with endothelial cells.

Primary cultures of Anaplasma marginale infected erythrocytes were used to determine conditions for in vitro cultivation of the rickettsia. The infected erythrocytes that were maintained by regular addition of Glasgow's MEM with fetal calf serum and uninfected erythrocytes showed a 1-5% increase in percent infected erythrocytes on the evaluation of Giemsa stained smears. This increase in parasitemia resulted in up to 70% change in the number of infected erythrocytes. Co-culture of the infected erythrocytes with endothelial cell monolayers allowed for longer maintenance with the parasitemia ranging from 5-13% through four passages over 16 weeks. Examination of cultures using transmission electron microscopy showed initial bodies within the erythrocytes at 10 days after the initial passage of the primary culture. The endothelial cell monolayers in the co-cultures contained multiple initial bodies. We have demonstrated that A. marginale can be grown for a limited number of passages in the co-culture system, which will facilitate the development of a continuous culture of the organism.

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.

Detection of Cowdria ruminantium by means of a DNA probe, pCS20 in infected bont ticks, Amblyomma hebraeum, the major vector of heartwater in southern Africa.

A DNA probe, pCS20, previously described for use in detection of Cowdria ruminantium infections in Amblyomma variegatum (the principal vector of heartwater) hybridized with C. ruminantium DNA in organs of laboratory-infected A. hebraeum adult ticks (the major southern African vector of heartwater). The probe hybridized with C. ruminantium DNA in 46/49 midguts from male ticks and 26/29 from females, thus indicating infection. Corresponding salivary glands were less heavily infected, but infections were more numerous in glands from males. Infection in ticks was confirmed by transmission of the disease to susceptible goats. The probe did not hybridize with DNA from uninfected ticks or with DNA from a spotted fever group rickettsia commonly associated with A. hebraeum in Zimbabwe. The C. ruminantium specific pCS20 DNA probe can be applied to determine accurately the infection rates in the two major vectors of heartwater and the risk of exposure of ruminants in endemic areas.

A cloned DNA probe for Cowdria ruminantium hybridizes with eight heartwater strains and detects infected sheep.

The DNA probe pCS20, which was cloned from the DNA of the Crystal Springs heartwater strain from Zimbabwe, cross-reacted with DNAs of heartwater strains from all endemic areas, including four heartwater strains from Zimbabwe, two strains from South Africa, one strain from Nigeria, and the Gardel strain from the Caribbean island of Guadeloupe. By nucleic acid hybridization, the pCS20 DNA probe detected Cowdria ruminantium DNA in all DNA preparations made from plasma samples from infected sheep before and during the febrile reaction. Synthetic oligonucleotides were prepared for amplification of specific C. ruminantium DNA sequences by the polymerase chain reaction (PCR). Amplification of two DNA products (181 and 279 bp) from pCS20 DNA and C. ruminantium genomic DNA of heartwater strains was demonstrated. In contrast, amplification of these products or any other products was not possible from genomic DNAs of Anaplasma marginale, Babesia bigemina, Trypanosoma brucei brucei, Escherichia coli, and bovine endothelial cells. The cross-reactivities of the 32P-labeled PCR products with genomic DNAs from several heartwater strains were similar to those with the pCS20 DNA probe. A nucleic acid-based test that uses hybridization assays and PCR provides a sensitive method for the detection of heartwater in both animals and ticks and has applications in epidemiological studies for the disease, which may allow for improved disease control.

A cloned DNA probe identifies Cowdria ruminantium in Amblyomma variegatum ticks.

Heartwater, caused by Cowdria ruminantium and transmitted by ticks of the genus Amblyomma, is a constraint to ruminant animal production in sub-Saharan Africa. This rickettsial disease could spread from endemically infected areas of sub-Saharan Africa and certain Caribbean islands to other countries, including the United States, in which Amblyomma ticks exist. To detect C. ruminantium in tick vectors and animals, we made DNA probes from C. ruminantium DNA isolated from endothelial cell cultures. Two clones were evaluated; pCS20 from Crystal Springs (Zimbabwe) strain DNA had a 1,306-bp insert, and pCR9 from Kiswani (Kenya) strain DNA had a 754-bp insert. Both DNA probes detected 1 ng of Crystal Springs DNA; however, the pCS20 probe had a 10-fold-greater ability to discriminate between C. ruminantium DNA and DNA from other organisms. Also, the pCS20 probe did not hybridize to 400 ng (highest amount tested) of DNA from bovine cells, 3 protozoa, 3 rickettsiae, and 12 bacteria. In all experiments, C. ruminantium DNA was detected in midguts from 99 of 160 Amblyomma variegatum nymphs infected as larvae and in midguts from 38 of 80 adult ticks infected as nymphs but not in midguts from control nymphs and adults. The presence of C. ruminantium in nymphs and adults was confirmed by transmission of heartwater to goats. The DNA sequences of both probes were determined; synthetic oligonucleotides from pCS20 are recommended as DNA probes for C. ruminantium.

Cloned DNA probes identify Anaplasma ovis in goats and reveal a high prevalence of infection.

Anaplasma organisms are observed in erythrocytes from goats with anemia and weight loss in Kenya. Three anaplasmas have been isolated in nature, Anaplasma ovis, Anaplasma marginale, and Anaplasma centrale. The two recognized species, A. ovis and A. marginale, are known to infect goats. Since only A. ovis causes clinical disease in goats, the Anaplasma species in goats in Kenya were identified. To detect A. ovis, a 9.6-kilobase-pair section of genomic DNA was cloned into pBR322 (pAO12A) and was used in conjunction with an A. marginale DNA probe previously derived from a gene coding for a 105,000-molecular-weight surface protein (Am105L) of A. marginale. In Southern blots, pAO12A DNA hybridized to several at least partially homologous sequences that were present in A. ovis and A. marginale genomic DNAs. The pAO12A DNA did not hybridize to Babesia bovis genomic or goat leukocyte DNA. The Anaplasma species that infected goats was identified as A. ovis by (i) DNA hybridization with pAO12A, (ii) hybridization of the A. marginale DNA probe to A. centrale and A. marginale genomic DNAs and lack of hybridization to A. ovis genomic DNA from an isolate obtained in Idaho and Anaplasma DNA from infected goats in Kenya, (iii) the intraerythrocytic location of Anaplasma organisms in infected goat blood, and (iv) the host specificity of the Anaplasma organisms for goats but not for cattle. Also, by using the two Anaplasma DNA probes, the prevalence of A. ovis in goats from seven locations in Kenya was found to range from 22 to 87%. The pAO12A DNA probe detected a 0.0035% A. ovis parasitemia in infected blood, an improved sensitivity which is suitable for use in surveillance and epidemiological studies.