Preliminary development of a polymerase chain reaction assay forAnaplasma marginale in ticks

Stich, Roger W.; Bantle, John A.; Kocan, Katherine M.; Eriks, Inge S.; Palmer, Guy H.

doi:10.1007/bf02438661

Cited by 5 publications

(6 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition, there are technical advantages of assaying tick vectors for rickettsiae. First, hemoglobin from the host bloodmeal does not appear to be a major problem with regard to inhibition of the PCR assay (33)(34)(35). Also, because invertebrate vectors usually are biological hosts for these pathogens, ticks can be used to biologically amplify the assay target organism prior to attempting PCR.…”

Section: Discussionmentioning

confidence: 99%

“…Conversely, PCR assays based on species-specific DNA sequences have met with success. For example, PCR has been utilized for amplification of msp-1␤ and msp5 sequences from the ehrlichial parasite Anaplasma marginale and resulted in highly sensitive and specific assays for that parasite (14,33,35). Thus, another approach to a PCR assay for E. canis would be to utilize a gene sequence that is unique to Ehrlichia spp.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Detection of Ehrlichia canis in Canine Carrier Blood and in Individual Experimentally Infected Ticks with a p30 -Based PCR Assay

et al. 2002

View full text Add to dashboard Cite

Detection of vector-borne pathogens is necessary for investigation of their association with vertebrate and invertebrate hosts. The ability to detect Ehrlichia spp. within individual experimentally infected ticks would be valuable for studies to evaluate the relative competence of different vector species and transmission scenarios. The purpose of this study was to develop a sensitive PCR assay based on oligonucleotide sequences from the unique Ehrlichia canis gene, p30, to facilitate studies that require monitoring this pathogen in canine and tick hosts during experimental transmission. Homologous sequences for Ehrlichia chaffeensis p28 were compared to sequences of primers derived from a sequence conserved among E. canis isolates. Criteria for primer selection included annealing scores, identity of the primers to homologous E. chaffeensis sequences, and the availability of similarly optimal primers that were nested within the target template sequence. The p30-based assay was at least 100-fold more sensitive than a previously reported nested 16S ribosomal DNA (rDNA)-based assay and did not amplify the 200-bp target amplicon from E. chaffeensis, the human granulocytic ehrlichiosis agent, or Ehrlichia muris DNA. The assay was used to detect E. canis in canine carrier blood and in experimentally infected Rhipicephalus sanguineus ticks. Optimized procedures for preparing tissues from these hosts for PCR assay are described. Our results indicated that this p30-based PCR assay will be useful for experimental investigations, that it has potential as a routine test, and that this approach to PCR assay design may be applicable to other pathogens that occur at low levels in affected hosts.Canine monocytic ehrlichiosis (CME), sometimes known as tropical canine pancytopenia, is a cosmopolitan tick-borne disease of dogs that is primarily caused by the rickettsia Ehrlichia canis (10,(16)(17)(18)31). This parasite invades and develops in monocytes and macrophages of dogs, resulting in leukopenia, thrombocytopenia, fever, depression, and anorexia, and this species has been reported in association with at least one human case (26). Three other ehrlichial organisms, E. chaffeensis, the human granulocytic ehrlichiosis (HGE) agent, and Ehrlichia ewingii, are zoonotic in the United States (5, 9), and several reports suggest that these organisms also occur naturally in dogs and deer (3, 4, 6-8, 11, 12, 15). Ixodid ticks transmit most of the Ehrlichia spp. characterized to date and appear to be the biological vectors of E. chaffeensis, the HGE agent, and E. ewingii. E. canis and E. chaffeensis are closely related, based on 16S rRNA gene (16S ribosomal DNA [rDNA]) sequences and antigenic cross-reactivity (2). These observations indicate that tick transmission and canine infection studies with E. canis can serve as a model for further work with E. chaffeensis in dogs and humans and lead to better understanding of both pathogens.A sensitive, specific PCR assay would be useful for detection of Ehrlichia spp. that typically occur at low l...

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Detection of Ehrlichia canis in Canine Carrier Blood and in Individual Experimentally Infected Ticks with a p30 -Based PCR Assay

et al. 2002

View full text Add to dashboard Cite

show abstract

“…29,30,31 The assay was found to be A. marginale-specific when tested with 18 species of bacteria and protozoa and 7 isolates of A. marginale from diverse geographical areas of the USA. 30 The data presented here have demonstrated the feasibility of using PCR to amplify and simultaneously label this 409-bp DNA fragment with digoxigenin-11-dUTP for development of a probe for detection of A. marginale.…”

Section: Discussionmentioning

confidence: 99%

“…The labeling reaction was more efficient than random primed DNA labeling, which has been the most efficient method reported previously. 8 As compared with standard PCR reaction conditions, 29,30,31 Recently, several radioactive nucleic acid probes have been developed for detection of A. marginale in bovine erythrocytes and tick tissues. These probes were constructed either from Anaplasma DNA genome 2, 35 or by recombinant techniques.…”

Section: Discussionmentioning

confidence: 99%

Detection ofAnaplasma MarginaleDNA in Bovine Erythrocytes by Slot-Blot and in Situ Hybridization with a PCR-Mediated Digoxigenin-Labeled DNA Probe

Kocan

Murphy

et al. 1995

J VET Diagn Invest

View full text Add to dashboard Cite

Abstract.A 409-base pair (bp) DNA fragment derived from the msp-1ß gene of Anaplasma marginale was amplified and simultaneously labeled with digoxigenin-11-dUTP by a polymerase chain reaction (PCR) assay. The resulting digoxigenin-labeled 409-bp PCR product was used as a probe for slot-blot and in situ hybridization to detect A. marginale DNA from experimentally infected bovine erythrocytes. The hybrid formation was detected with alkaline phosphatase-conjugated anti-digoxigenin antibody and substrates 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt. In slot-blot hybridizations, the probe detected A. marginale DNA from approximately 1,000-10,000 infected erythrocytes in 1.25 ml of whole blood, which is equivalent to a parasitemia level of 0.00001%. The probe proved to be A. marginale-specific when tested with 17 species of microorganisms. The applicability of the probe for diagnosis was tested by screening A. marginale infections in 2 experimentally infected splenectomized cattle before microscopically detectable parasitemias and after acute infection. After inoculation of infected blood, A. marginale infections were detected with the probe 14 days prior to detection in stained smears. Microscopically inapparent parasitemias were also detected with the probe for 2 months after acute disease. When the probe was used for in situ hybridization on methanol-fixed blood smears, probe reaction could be visualized with light microscopy on A. marginale inclusions within infected erythrocytes. The probe reaction was not observed on leukocytes and uninfected erythrocytes from infected blood smears, on erythrocytes from uninfected blood samples, or on samples infected with A. ovis, Babesia bovis, or B. bigemina. This PCR-mediated nonradioactive probe appears to be a sensitive diagnostic test for A. marginale.Anaplasmosis is a tick-borne disease of cattle and other ruminants caused by the intraerythrocytic rickettsia, Anaplasma marginale. The disease is the most prevalent of bovine hemoparasite infections and is enzootic to nearly half the world's livestock production regions. 23 Invasion and multiplication of A. marginale in erythrocytes of cattle may result in anemia, weight loss, abortion, and sometimes death during acute infections. 26 Cattle recovering from acute disease remain persistent carriers with low to inapparent parasitemias and may serve as reservoirs for transmission of the organism. 32 The annual losses attributed to anaplasmosis within the United States alone were estimated at $300 million. 23 Diagnosis of acute anaplasmosis is often based on direct microscopic detection of Anaplasma inclusions in Giemsa-stained blood smears. 28 Infected erythrocytes in carrier cattle are often not detectable by microscopic examination of stained blood smears (less than 0.1% infected erythrocytes). 18 28 and radioimmunoassay. These tests often lack sensitivity and specificity, or some of them do not distinguish infected from uninfected vaccinated cattle. Detection of A. marginale using nucleic acid h...

show abstract

“…34 This fragment was used previously in our laboratory for development of a PCR assay for detection of A. marginale in hemolymph, oral secretions, midgut, and salivary glands of experimentally infected ticks. 35‐37 The PCR assay was found to be A. marginale ‐specific when tested with 18 species of bacteria and protozoa and 7 isolates of A. marginale from diverse geographical areas of the USA. 35…”

Section: Development Of a Non‐radioactive Dna Probe For Detection Of mentioning

confidence: 99%

Development of a Non‐radioactive DNA Probe andin SituHybridization for Detection ofAnaplasma marginalein Ticks and Cattle^a

Kocan

Blouin

et al. 1998

Annals of the New York Academy of Sciences

View full text Add to dashboard Cite

A non-radioactive DNA probe was developed for detection of Anaplasma marginale in ticks and cattle. The probe was labeled with digoxigenin 11-dUTP by polymerase chain reaction. The probe was tested on bovine blood and was found to be a sensitive and specific detection method for A. marginale in cattle. The DNA probe was then adapted for in situ hybridization (ISH) of A. marginale in Dermacentor andersoni and D. variabilis ticks infected either as nymphs or adults. One-half of each tick was studied with ISH while the other half was examined with light and electron microscopy. In male ticks infected as adults, tick gut cells first became infected with A. marginale while ticks fed on an infected calf, and they remained infected as they transmission fed on a second, susceptible calf. At the onset of transmission feeding, salivary glands became infected with A. marginale. During transmission feeding infection was also observed in interstitial, reproductive, skeletal muscle, fat body and Malpighian tubule tissue, resulting in a generalized A. marginale infection. When adult ticks that acquired infection as nymphs were examined with ISH and microscopy, gut tissues of both D. andersoni and D. variabilis became infected with A. marginale. However, salivary gland infection was seen only in D. variabilis, even though both species of ticks transmitted A. marginale to susceptible calves. A. marginale was not seen with ISH or microscopy in hemocytes collected from both species of ticks and, thus, hemocytes do not appear to play a role in the development of A. marginale in ticks.

show abstract

Preliminary development of a polymerase chain reaction assay forAnaplasma marginale in ticks

Cited by 5 publications

References 8 publications

Detection of Ehrlichia canis in Canine Carrier Blood and in Individual Experimentally Infected Ticks with a p30 -Based PCR Assay

Detection of Ehrlichia canis in Canine Carrier Blood and in Individual Experimentally Infected Ticks with a p30 -Based PCR Assay

Detection ofAnaplasma MarginaleDNA in Bovine Erythrocytes by Slot-Blot and in Situ Hybridization with a PCR-Mediated Digoxigenin-Labeled DNA Probe

Development of a Non‐radioactive DNA Probe andin SituHybridization for Detection ofAnaplasma marginalein Ticks and Cattle^a

Contact Info

Product

Resources

About

Preliminary development of a polymerase chain reaction assay forAnaplasma marginale in ticks

Cited by 5 publications

References 8 publications

Detection of Ehrlichia canis in Canine Carrier Blood and in Individual Experimentally Infected Ticks with a p30 -Based PCR Assay

Detection of Ehrlichia canis in Canine Carrier Blood and in Individual Experimentally Infected Ticks with a p30 -Based PCR Assay

Detection ofAnaplasma MarginaleDNA in Bovine Erythrocytes by Slot-Blot and in Situ Hybridization with a PCR-Mediated Digoxigenin-Labeled DNA Probe

Development of a Non‐radioactive DNA Probe andin SituHybridization for Detection ofAnaplasma marginalein Ticks and Cattlea

Contact Info

Product

Resources

About

Development of a Non‐radioactive DNA Probe andin SituHybridization for Detection ofAnaplasma marginalein Ticks and Cattle^a