PubMed Health⌕ Search

Biomedical subjects

K M Kocan

Publications and source records attributed to K M Kocan.

At least 55 records · Page 3Linked to original sources
PubMed1997

Use of a nonradioactive DNA probe for detection of Anaplasma marginale infection in field cattle: comparison with complement fixation serology and microscopic examination.

A sensitive Anaplasma marginale-specific 409-base pair DNA probe was developed in a previous study for detection of A. marginale infection in experimentally infected cattle with a test that employed slot-blot and in situ hybridization. To test the suitability of the probe to detect A. marginale in the blood of naturally infected carrier cattle, slot-blot hybridization was used to determine the infection rate of A. marginale in cattle from 3 geographic areas in Oklahoma. For comparison, blood samples from the same cattle were also examined by light microscopy and were tested by the complement fixation test. For the DNA hybridization assay, the probe was labeled with digoxigenin 11-dUTP by polymerase chain reaction (PCR). DNA was extracted from blood using the QIAamp blood kit and then applied to a nylon membrane and hybridized with the probe. The study herds consisted of 31 beef cows in Harper County, OK, and 42 and 70 dairy cows from Payne and Pittsburg counties, OK, respectively. In the 3 herds, 80.6%, 92.8%, and 57.1% of the cows were positive for A. marginale as assessed with the DNA hybridization assay. In contrast, only 25.8% and 2.86% were complement fixation positive in 2 herds, and no complement fixation positives were found in 1 herd. Uncountable parasitemia that was too low to accurately determine (< 0.01%) from 29.0%, 4.8%, and 11.4% of the samples, respectively, was demonstrated by microscopic examination. All samples positive by complement fixation and microscopic examination had positive probe reactions in the DNA hybridization assay. Therefore, the PCR-mediated nonradioactive DNA probe described here may be useful in epidemiologic investigations and in identification of carrier cattle. This assay could be adapted for use in diagnostic laboratories because it is sensitive, specific, nontoxic, quickly executed, and inexpensive.

Anaplasma↗
PubMed1996

Strategies to interrupt the development of Anaplasma marginale in its tick vector. The effect of bovine-derived antibodies.

Anaplasma marginale is a rickettsia transmitted by ticks that invades and multiplies in bovine erythrocytes causing the disease anaplasmosis. A complex developmental cycle occurs within ticks that begins in midgut cells, with subsequent infection in gut muscle cells. Final development occurs in salivary glands from where the rickettsia is transmitted to the vertebrate host. At each site of development, A. marginale multiplies within membrane-bound inclusions. Attempts to control anaplasmosis have focused on cattle and have included immunization and prophylactic treatment with tetracyclines. New strategies for control of anaplasmosis are being focused on the tick vector. Development of vaccines against hemoparasites in ticks may be feasible because vertebrate host immunoglobulins appear to cross the midgut epithelium of invertebrates and enter the hemolymph without breakdown. We tested the effect of A. marginale antibodies ingested by ticks with the bloodmeal on infections in ticks. Cattle were immunized with purified outer membrane proteins of erythrocytic-derived parasites. Infections in ticks exposed to the immunized cattle were determined using an Anaplasma-specific DNA probe, light and electron microscopy, and tick transmission studies. Vaccine-derived antibodies did not appear to affect the development and transmission of A. marginale in ticks. Further studies are needed to determine if bovine antibodies remain intact within ticks and whether the tick stage of A. marginale has unique surface antigens from the erythrocytic stage.

Anaplasma↗
PubMed1996

Preliminary studies on the effect of Anaplasma marginale antibodies ingested by Dermacentor andersoni ticks (Acari:Ixodidae) with their blood meal on infections in salivary glands.

The effect of Anaplasma marginale antibodies ingested with the tick blood meal was tested on infected male ticks that were allowed to feed on cattle immunized with the erythrocytic stage of A. marginale. The experiments were done in two trials. Trial 1 was done using splenectomized calves (two calves per treated and control groups) while ticks in trial 2 were fed on intact yearling cattle (four cattle per treated and control groups). The cattle were immunized with purified outer membrane proteins of erythrocyte-derived A. marginale using saponin (trial 1) or monophosphoryl lipid-A-trehalose dicorynomycolate adjuvant (trial 2). The corresponding control cattle received adjuvant only. All cattle were challenged using Dermacentor andersoni males infected as adults that were allowed to feed for 7 days. In trial 1, the ticks were allowed to feed a second time on susceptible calves to test whether exposure of ticks to immunized cattle affected their ability to transmit anaplasmosis. Infections in fed ticks were monitored by determining the infection rates in salivary glands with an A. marginale-specific RNA probe and light microscopy. Vaccine-derived antibodies ingested with the tick blood meal did not appear to affect the development of A. marginale in previously infected ticks. The infection rates in the salivary glands were not significantly different among ticks fed on immunized versus adjuvant control cattle. When the vaccine-exposed ticks in trial 1 were allowed to feed a second time on susceptible calves, the resulting clinical symptoms of anaplasmosis were similar to those of the controls. There was no statistically significant effect of tick exposure to the anti-erythrocytic stage antibody on the development of salivary gland infection or transmission of A. marginale by ticks.

Anaplasma↗
PubMed1996

Establishment of the tick (Acari:Ixodidae)-borne cattle pathogen Anaplasma marginale (Rickettsiales:Anaplasmataceae) in tick cell culture.

Anaplasma marginale is a tick-borne rickettsia that causes bovine anaplasmosis worldwide. Despite its importance, A. marginale has thus far not been established in a continuous culture system. We have propagated A. marginale continuously for the 1st time in a tick cell line derived from the black-legged tick, Ixodes scapularis Say, using infected bovine blood as the inoculum. Erythrocytic stages invaded the tick cells and multiplied in membrane-lined vacuoles to form colonies typical of those observed in naturally infected ticks as demonstrated by light and electron microscopy. The rickettsiae have been passaged serially for 3 yr and have been cryopreserved in liquid nitrogen. Antigens present in A. marginale from tick cell culture were recognized by bovine immune serum against the blood stages of A. marginale. A. marginale grown in this tick cell line was infective for calves, and male ticks fed on the calves transmitted A. marginale to a susceptible calf. The ability to culture A. marginale removes a major impediment to the study of Anaplasma biology in vitro, and will enhance development of vaccines and diagnostic tests.

Anaplasma↗
PubMed1996

Developmental studies of Anaplasma marginale (Rickettsiales:Anaplasmataceae) in male Dermacentor andersoni (Acari:Ixodidae) infected as adults by using nonradioactive in situ hybridization and microscopy.

The development of Anaplasma marginale Theiler was studied in ticks using a nonradioactive in situ hybridization method developed in our laboratory. Male Rocky Mountain wood ticks, Dermacentor andersoni Stiles, were infected intrastadially by allowing them to feed for 7 d on an infected calf (acquisition feeding). The ticks were then removed and held in a humidity chamber for 5 d before being fed on a 2nd susceptible call for 10 d (transmission feeding). Two groups of 10 ticks were collected daily during the 22-d experiment. In one group one-half of each tick was processed and embedded in paraffin and in the other group one-half of each tick was embedded in LR White for in situ hybridization. The companion tick halves from each group were fixed and embedded in Dow Epoxy Resin resin for routine light and electron microscopy. As detected by in situ hybridization on LR White- and paraffin-embedded sections and by microscopy, initial infection of A. marginale in ticks occurred in gut tissues either on the 7th d of acquisition feeding or the 1st d of the held period and infection persisted throughout transmission feeding. The highest number of ticks with gut infection was observed on the 5th d of transmission feeding. Salivary glands became infected with A. marginale on the 1st day of transmission feeding and remained infected throughout the transmission feeding period. Peak infection was observed on day 4 of transmission feeding. After the beginning of transmission feeding, A. marginale infection was also observed in interstitial, reproductive, skeletal muscle, fat body, and Malpighian tubule tissues. Although A. marginale infection of ticks clearly originates in midgut epithelial cells, many tissues eventually become infected during transmission feeding, resulting in a generalized infection. The infection of multiple tissues may contribute to the ability of A. marginale infection to persist in intrastadially infected male ticks.

Anaplasma↗
PubMed1996

Attempted transmission of Ehrlichia canis by Rhipicephalus sanguineus after passage in cell culture.

OBJECTIVES: To compare the transmissibility by the brown dog tick, Rhipicephalus sanguineus, of a recent isolate of Ehrlichia canis (Ebony) with that of another isolate (Oklahoma) that had been passaged in cell culture, and to assess the genetic similarity of the 2 isolates as reflected in the nucleotide (NT) sequence of 16S rDNA. ANIMALS: 13 healthy dogs of various ages and breeds. PROCEDURE: Larval and nymphal ticks were acquisition fed on acutely infected dogs, and, after molting, they were transmission fed as nymphs and adults, respectively, on Ehrlichia-naive dogs. All dogs were monitored daily by blood smear evaluation for evidence of parasitized leukocytes and by physical examination for clinical signs of ehrlichiosis. Serologic and hematologic values were measured weekly. Using a nested polymerase chain reaction, the 16S rDNA was amplified, and the NT sequence of the template DNA was determined. RESULTS: The Ebony isolate of E canis was successfully transmitted to dogs by nymphal and adult ticks. In contrast, no ticks that fed on dogs harboring the cell-cultured isolate (Oklahoma) transmitted it to dogs. On the basis of 16S rDNA sequence, the 2 isolates were 99.9% similar, with only 1 NT difference. CONCLUSIONS: These results reconfirm the vector potential of R sanguineus for E canis. Passage of the Oklahoma isolate of E canis in cell culture apparently adversely affected its transmissibility by ticks, raising the possibility that cell-cultured isolates of this rickettsia may lose their affinity for ticks. Determination of 16S rDNA sequence suggests minor strain variation within the species E canis.

Animals↗
PubMed1995

Targeting ticks for control of selected hemoparasitic diseases of cattle.

Development in and transmission of hemoparasites by tick vectors are phenomena closely synchronized with the tick feeding cycle. In all known life cycles, initial infection of tick tissues occurs in midgut epithelial cells and transmission is effected as ticks feed after parasites have developed and multiplied in salivary glands. Many factors reviewed affect development and transmission of hemoparasites by ticks including age of ticks, artificial temperature, climate and/or season, tick stage or sex, hemoparasite variation, concurrent infection of ticks with other pathogens, host cell susceptibility, transovarial transmission, effect of hemoparasites on tick biology, and the effect of infecting parasitemia level in cattle on infection rates in ticks. Four hemoparasites of cattle, Anaplasma marginale, Cowdria ruminantium, Theileria parva, and Babesia spp., are all dependent on ticks for biological transmission. Babesia is transmitted transovarially whereas the other three are transmitted transstadially. Mechanical transfer of infective blood via fomites and mouthparts of biting arthropods is also a major means of transmission for Anaplasma marginale but not of the others. Potential control methods for hemoparasites that target parasites as they are developing in their respective tick hosts include tick control, vaccines (against ticks and parasites), and drugs (against ticks and parasites). Successful application of control strategies will be dependent upon thorough understanding of parasite developmental cycles, biology of the tick vectors and the immune response of cattle to ticks and to hemoparasites. The most effective control measures will be those that are targeted against both ticks and the hemoparasites they vector.

Animals↗
PubMed1995

Experimental transmission of Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) among white-tailed deer by Amblyomma americanum (Acari: Ixodidae).

Ehrlichia chaffeensis Anderson, Dawson & Wilson, causative agent of human (predominantly monocytic) ehrlichiosis, was successfully transmitted experimentally by Amblyomma americanum (L.) to white-tailed deer, Odocoileus virginianus (Zimmerman). Deer were needle-exposed intravenously to E. chaffeensis in tissue-culture canine macrophage (DH82) cells, and 11 d later were exposed to laboratory-reared A. americanum larvae, nymphs, and adults for acquisition feeding. Three months after this feeding, naive deer and dogs were exposed to recently molted nymphs and adults. Attempted reisolation of the pathogen by way of tissue culture was successful from one needle-exposed deer but not from the tick-exposed deer or dogs. Based on serologic evidence and polymerase chain reaction data, both nymphal and adult ticks transmitted E. chaffeensis to naive deer but not to dogs.

Animals↗
PubMed1995

Detection of Anaplasma marginale DNA in bovine erythrocytes by slot-blot and in situ hybridization with a PCR-mediated digoxigenin-labeled DNA probe.

A 409-base pair (bp) DNA fragment derived from the msp-1 beta 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 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.

Anaplasma↗
PubMed1993

Detection of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in hemolymph of Dermacentor andersoni (Acari: Ixodidae) with the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to detect Anaplasma marginale in hemolymph collected from live Dermacentor andersoni Stiles ticks. Hemolymph was collected from severed legs of male and female ticks exposed to A. marginale as either nymphs or adults. Heat treatment was found to be the optimum method of hemolymph preparation for PCR. Hemolymph samples were collected and pooled from adult ticks exposed as nymphs on days 0-10 of feeding on a susceptible calf. For male and female ticks exposed as adults, samples were collected as ticks fed 7 d on an infected calf, while being held 9 d between feedings, and during a second feeding of 10 d (or to repletion) when they transmitted the parasite. Hemolymph samples were collected from uninfected ticks at the same times to serve as controls. Anaplasma marginale DNA was amplified with primers BAP-2 (5'-GTATGGCACGTAGTCTTGGGATCA-3') and AL34S (5'-CAGCAGCAGCAAGACCTTCA-3'), which flank a 409-bp fragment of the A. marginale Florida isolate msp1 beta gene. Infected tick hemolymph was PCR-positive for A. marginale at all collection times, including unfed adults infected as nymphs and previously unexposed adults that fed on infected calves for only 1 d. The PCR-based assay of tick hemolymph proved to be a sensitive method for identification of infected ticks, potentially without killing them; it would be well suited for identification of laboratory- or field-infected ticks that could then be used for further studies. The primers used in this assay were also found specific when tested with species of 18 different genera, and universal for 7 A. marginale isolates from diverse geographical areas of the United States.

Anaplasma↗
PubMed1993

Detection of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in secretagogue-induced oral secretions of Dermacentor andersoni (Acari: Ixodidae) with the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to detect Anaplasma marginale in secretagogue-induced oral secretions of male and female Dermacentor andersoni Stiles exposed as nymphs or adults by feeding on infected calves. A 409-bp DNA fragment derived from the A. marginale (Florida isolate) msp1 beta gene was amplified with oligonucleotide primers BAP-2 (5'-GTATGGCACGTAGTCTTGGGATCA-3') and AL34S (5'-CAGCAGCAGCAAGACCTTCA-3'). The target DNA was amplified in oral secretions of female ticks exposed to A. marginale as adults and stimulated to secrete by injection of dopamine. Conversely, A. marginale was detected in saliva from prefed female ticks exposed as nymphs only after stimulation with a combination of dopamine, gamma-aminobutyric acid, pilocarpine, and theophylline. Saliva from ticks exposed as nymphs and stimulated with ergot alkaloids did not contain the A. marginale target DNA. Saliva collected after 11 d of feeding from dopamine-stimulated male ticks contained A. marginale DNA. The results indicate that A. marginale is present in tick saliva and suggest that the parasite can be transmitted to cattle via saliva of feeding ixodid ticks. The variable appearance of A. marginale in saliva, regardless of the method used to induce salivation, suggests that transmission of A. marginale may be affected by the physiological state of the tick.

Anaplasma↗
PubMed1993

Development and transmission of Cowdria ruminantium by Amblyomma males transferred from infected to susceptible sheep.

Male Amblyomma sp. were tested as vectors of Cowdria ruminantium, causative agent of heartwater disease. The males were allowed to feed on sheep experimentally infected with C. ruminantium and then were transferred to susceptible sheep to test for transmission of the rickettsia. The experiments were done in two trials. In the first trial, A. hebraeum were exposed to the Palm River stock of C. ruminantium, while in the second trial the Kiswani stock of Cowdria was tested with A. variegatum. Ticks were collected daily throughout each experiment, cut in half, and processed for light and electron microscopy to study development of C. ruminantium in tick tissues. In both trials, the male ticks transmitted Cowdria to one of two susceptible sheep. When ticks were examined with microscopy, a few colonies were found in gut cells while none were seen in salivary glands. Both species of ticks were infected with Rickettsia conorii, as evidenced by the occurrence of rickettsiae in the nucleus and cytoplasm of salivary gland cells.

Animals↗
PubMed1993

Persistence of tick-derived Anaplasma marginale in cultured bovine turbinate and endothelial cells.

Anaplasma marginale from salivary glands of Dermacentor andersoni was used to inoculate monolayers of bovine turbinate and endothelial cells. Monolayers were passaged at 2 or 4 week intervals and monitored with light and electron microscopy and with an A. marginale-specific DNA probe. Intracellular inclusions were observed in turbinate cells after 2-4 weeks. The number of inclusion-bearing cells increased over 1-2 weeks and gradually disappeared. A radiolabeled fragment from within the msp1 beta gene of the erythrocytic stage of A. marginale hybridized to DNA extracted from bovine turbinate cell cultures as late as 7 weeks post inoculation (passage 4). Individual rickettsiae were observed with electron microscopy in samples taken at this time. Susceptible calves inoculated with suspect cultures did not develop clinical anaplasmosis but did develop significant antibody titers as detected with ELISA. DNA from endothelial cell cultures at 9 weeks post inoculation also bound the Anaplasma-specific DNA probe. Anaplasma marginale from salivary glands of D. andersoni appears to persist in cultured bovine turbinate and endothelial cells but typical development and infectivity for bovines do not occur.

Anaplasma↗
PubMed1993

Effect of 4-bromo-calcium ionophore A23187 on release of Anaplasma marginale from bovine erythrocytes in vitro.

The ionophore A23187 was used to facilitate release and continued development of Anaplasma marginale in short-term erythrocyte cultures. Addition of 10 microM A23187 to the cultures resulted in significant decrease in percentage of parasitized erythrocytes (PPE) by 24 hours after treatment; further development and increase in PPE was not observed. In contrast, the PPE of untreated cultures, those treated with dimethyl sulfoxide (DMSO) only and with 1 microM A23187 increased slightly during that time. Total erythrocyte count decreased in treated cultures in excess of that expected after samples of the medium were taken for analysis. The greatest cell loss and increased hemoglobin concentration in culture medium was observed in cultures treated with 10 microM A23187 and with an equivalent volume of DMSO. The DMSO appeared to cause hemolysis of some erythrocytes, but not of infected cells selectively. Release of A marginale inclusion bodies was seen by electron microscopy in samples from the 10 microM A23187-exposed cultures. At 30 minutes after treatment, free initial bodies were frequently seen. Inclusion body membranes and individual A marginale were associated with membranes of adjacent erythrocytes. Individual rickettsiae were seen in cell depressions and appeared to be entering erythrocytes. However, neither further invasion nor development of the parasite in erythrocytes was observed. Ionophore A23187 appeared to promote release of A marginale from erythrocytes, but did not enhance infection of erythrocytes or development of organisms in vitro.

Anaplasma↗
PubMed1993

Development of Anaplasma marginale in salivary glands of male Dermacentor andersoni.

Development of the rickettsia, Anaplasma marginale, in salivary glands of male Dermacentor andersoni exposed as nymphs or adult ticks, was studied indirectly by inoculation of susceptible calves with homogenates and directly by examination, using light microscopy and a DNA probe; some unfed ticks were incubated before tissues were collected. Salivary gland homogenates made from ticks in every treatment group caused anaplasmosis when injected into susceptible calves; prepatent periods decreased as the time that ticks had fed increased. Colonies of A marginale were seen only in salivary glands of ticks exposed as adults and not in those exposed as nymphs; the percentage of salivary gland acini infected in these ticks increased linearly with feeding time. However, the probe detected A marginale DNA in salivary glands of ticks from both groups; the amount of DNA detected increased as feeding time was extended. The amount of A marginale DNA appeared to remain constant in gut tissues, but to increase in salivary glands. Salivary glands of adult-infected male ticks that were incubated, but did not feed a second time, became infected with A marginale, and the pattern of infection of acini varied with incubation temperature. Development of A marginale in salivary glands appears to be coordinated with the tick feeding cycle; highest infection rate was observed in ticks exposed as adults.

Anaplasma↗