Papers by Katherine Kocan
PLOS ONE, 2015
Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that ... more Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10-15% and 65-71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands PLOS ONE | training network for capacity building to control ticks when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophiluminfected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface proteins during A. phagocytophilum infection in ticks. Characterization of Anaplasma proteome contributes information on host-pathogen interactions and provides targets for development of novel control strategies for pathogen infection and transmission.
Anaplasma marginale, a tick-borne rickettsial pathogen of cattle, infects bovine erythrocytes, re... more Anaplasma marginale, a tick-borne rickettsial pathogen of cattle, infects bovine erythrocytes, resulting in mild to severe hemolytic disease that causes economic losses in domestic livestock worldwide. Recently, the Virginia isolate of A. marginale was propagated in a continuous tick cell line, IDE8, derived from embryonic Ixodes scapularis. Development of A. marginale in cell culture was morphologically similar to that described
PloS one, 2015
Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basi... more Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basic RNA interference (RNAi) machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor-SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined ...
Veterinary parasitology, Jan 10, 2010
The intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), described by Sir... more The intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), described by Sir Arnold Theiler in 1910, is endemic worldwide in tropical and subtropical areas. Infection of cattle with A. marginale causes bovine anaplasmosis, a mild to severe hemolytic disease that results in considerable economic loss to both dairy and beef industries. Transmission of A. marginale to cattle occurs biologically by ticks and mechanically by biting flies and by blood-contaminated fomites. Both male ticks and cattle hosts become persistently infected with A. marginale and serve as reservoirs of infection. While erythrocytes are the major site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks that begins by infection of gut cells, and transmission to susceptible hosts occurs from salivary glands during feeding. Major surface proteins (MSPs) play a crucial role in the interaction of A. marginale with host cells, and include adhesion proteins and MSPs ...
Parasitology research, 2007
RNA interference (RNAi) has become the most powerful experimental tool for the study of gene func... more RNA interference (RNAi) has become the most powerful experimental tool for the study of gene function in ticks. Subolesin, initially called 4D8, was found to be protective against tick infestations when used as a vaccine and was shown to be highly conserved among ixodid tick species at the nucleotide and protein levels. RNAi caused systemic silencing of subolesin and demonstrated that this protein is involved in regulation of tick feeding, reproduction, and development. Recently, these results were extended to the one-host tick Rhipicephalus (Boophilus) microplus in which injection of dsRNA into replete females resulted in transovarial silencing of subolesin expression in eggs and larvae. Herein, we report transovarial silencing of subolesin by RNAi in the three-host ticks, Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis. Silencing of subolesin expression by RNAi in these tick species also affected subolesin expression in eggs and larvae. Transovarial RNAi appear...
Trends in parasitology, 2006
Tick control is a subject that has stimulated intense interest for more than a century. This arti... more Tick control is a subject that has stimulated intense interest for more than a century. This article is a commentary on the research needs for tick control proposed in Peter Willadsen's recent article and it calls attention to tick control strategies that were either poorly represented or omitted from the latter. Special consideration is given to host-targeted devices to control disease vector ticks infesting wildlife, to pheromone-impregnated decoys for attracting and killing ticks in the natural environment and on hosts, and to more up-to-date advances in vaccine development.
BMC veterinary research, 2006
The genetic diversity of Anaplasma platys (Rickettsiales: Anaplasmataceae) strains is currently p... more The genetic diversity of Anaplasma platys (Rickettsiales: Anaplasmataceae) strains is currently poorly defined. The present study was designed to characterize A. patys strains in dogs from Palermo, Sicily, Italy, using a combination of PCR and sequence analysis of the 16S rDNA, heat shock operon groESL and citrate synthase (gltA) genes. Blood was collected from 344 dogs (111 pet dogs, 122 pound dogs and 111 hunting dogs) during 2003-2005 in the Province of Palermo, Sicily, Italy. The prevalence of A. platys in dogs in Sicily, as demonstrated by PCR and sequence analysis of the 16S rDNA, groESL and gltA genes, was 4%. None of the samples were positive for A. marginale, A. centrale, A. ovis and A. phagocytophilum DNA. Three different gltA genotypes of A. platys were identified in dogs from Sicily. Two of the gltA sequences of Sicilian A. platys strains were different from sequences reported previously. However, one of the gltA, 16S rDNA and groESL sequences were identical to the seque...
Annals of clinical microbiology and antimicrobials, 2005
Anaplasma phagocytophilum causes human granulocytic anaplasmosis (HGA) in humans, which has been ... more Anaplasma phagocytophilum causes human granulocytic anaplasmosis (HGA) in humans, which has been recognized as an emerging tick-borne disease in the United States and Europe. Although about 65 cases of HGA have been reported in Europe, some of them do not fulfill the criteria for confirmed HGA. Confirmation of HGA requires A. phagocytophilum isolation from blood, and/or identification of morulae in granulocytes and/or positive PCR results with subsequent sequencing of the amplicons to demonstrate specific rickettsial DNA. Seroconversion or at least fourfold increase in antibody titers to A. phagocytophilum has been used as criteria for confirmed HGA also. Infection with A. phagocytophilum was confirmed by PCR in a patient in Sicily, Italy, who had negative serology for A. phagocytophilum. A fragment of A. phagocytophilum 16S rDNA was amplified by two independent laboratories and sequenced from two separate patient's blood samples. The 16S rDNA sequence was identical in both samp...
Veterinary parasitology, Jan 9, 2001
Anaplasma marginale (A. marginale) is a tick-borne ehrlichial pathogen of cattle that causes the ... more Anaplasma marginale (A. marginale) is a tick-borne ehrlichial pathogen of cattle that causes the disease anaplasmosis. Six major surface proteins (MSPs) have been identified on A. marginale from cattle and ticks of which three, MSP1a, MSP4 and MSP5, are from single genes and do not vary within isolates. The other three, MSP1b, MSP2 and MSP3, are from multigene families and may vary antigenically in persistently infected cattle. Several geographic isolates have been identified in the United States which differ in morphology, protein sequence and antigenic properties. An identifying characteristic of A. marginale isolates is the molecular weight of MSP1a which varies in size among isolates due to different numbers of tandemly repeated 28-29 amino acid peptides. For these studies, genes coding for A. marginale MSP1a and MSP4, msp1alpha and msp4, respectively, from nine North American isolates were sequenced for phylogenetic analysis. The phylogenetic analysis strongly supports the exis...
Parasitology today (Personal ed.), 1999
Suman Mahan and co-authors review the strategies applied to develop improved vaccines for Cowdria... more Suman Mahan and co-authors review the strategies applied to develop improved vaccines for Cowdria ruminantium infections (heartwater). Inactivated vaccines using cell-cultured C. ruminantium organisms combined with an adjuvant are capable of protecting goats, sheep and cattle against lethal C. ruminantium challenge. Immune responses induced with this vaccine, or after recovery from infection, target outer membrane proteins of C. ruminantium, in particular the major antigenic protein 1 (MAP-1). Genetic immunizations with the gene encoding MAP-1 induce protective T helper cell type 1 responses against lethal challenge in a mouse model. Similarly, homologues of MAP-1 in other phylogenetically and antigenically related ehrlichial agents such as Anaplasma marginale and Ehrlichia chaffeensis are also targets of protective responses. Given the antigenic similarities between the related ehrlichial agents, common strategies of vaccine development could be applied against these agents that ca...
Infection and immunity, 1998
Major surface proteins of Anaplasma marginale are vaccine candidates. We recently demonstrated th... more Major surface proteins of Anaplasma marginale are vaccine candidates. We recently demonstrated that immunization of calves with outer membranes of the Florida strain of A. marginale resulted in protective immunity that correlated with a memory CD4(+) T-lymphocyte response specific for major surface protein 1 (MSP-1), MSP-2, and MSP-3 (W. C. Brown, V. Shkap, D. Zhu, T. C. McGuire, W. Tuo, T. F. McElwain, and G. H. Palmer, Infect. Immun. 66:5406-5413, 1998). As immunogens, these proteins have been shown to induce complete or partial protection against homologous challenge. To further define the T helper (Th) cell response to these and other A. marginale antigens and to determine conservation of Th cell epitopes among genetically distinct A. marginale strains, Th cell clones obtained prior to challenge from three immunized calves were characterized for antigen-specific responses. Nine distinct antigenic profiles were defined by 11 Th cell clones derived by stimulation with the Florida ...
PLOS Genetics, 2015
Anaplasma phagocytophilum is an emerging pathogen that causes human granulocytic anaplasmosis. In... more Anaplasma phagocytophilum is an emerging pathogen that causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects cell function in both vertebrate host and the tick vector, Ixodes scapularis. Global tissue-specific response and apoptosis signaling pathways were characterized in I. scapularis nymphs and adult female midguts and salivary glands infected with A. phagocytophilum using a systems biology approach combining transcriptomics and proteomics. Apoptosis was selected for pathway-focused analysis due to its role in bacterial infection of tick cells. The results showed tissue-specific differences in tick response to infection and revealed differentiated regulation of apoptosis pathways. The impact of bacterial infection was more pronounced in tick nymphs and midguts than in salivary glands, probably reflecting bacterial developmental cycle. All apoptosis pathways described in other organisms were identified in I. scapularis, except for the absence of the Perforin ortholog. Functional characterization using RNA interference showed that Porin knockdown significantly increases tick colonization by A. phagocytophilum. Infection with A. phagocytophilum produced complex tissue-specific alterations in transcript and protein levels. In tick nymphs, the results suggested a possible effect of bacterial infection on the inhibition of tick immune response. In tick midguts, the results suggested that A. phagocytophilum infection inhibited cell apoptosis to facilitate and establish infection through up-regulation of the JAK/STAT pathway. Bacterial infection inhibited the intrinsic apoptosis pathway in tick salivary glands by down-regulating Porin expression that resulted in the inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection. However, tick salivary glands may promote apoptosis to limit bacterial infection through induction of the extrinsic apoptosis pathway. These dynamic changes in response to A. phagocytophilum in I. scapularis tissue-specific transcriptome and proteome demonstrated the complexity of the tick response to infection and will contribute to characterize gene regulation in ticks.
Veterinary Microbiology, 2003
The major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: ... more The major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: Anaplasmataceae) has been shown to mediate adhesion, infection and transmission of the organism, as well as to contribute to protective immunity in cattle. MSP1a contains a variable number of tandemly repeated peptides in the amino-terminal region, while the remainder of the protein is highly conserved among isolates. The number of repeats varies among geographic isolates of A. marginale but is constant within an isolate and has been used as a stable genetic marker of isolate identity. Because the sequence of the tandem repeats is the most variable part of the protein among isolates, this region of the protein is most likely to be involved in adhesion to host cells, a prerequisite to infection. The purpose of this study was to characterize the organization and function of the MSP1a tandem repeats of A. marginale in adhesion to host cells. We demonstrated by use of recombinant mutant proteins that the tandemly repeated region of MSP1a was necessary and sufficient to mediate adhesion of MSP1a to tick cells and bovine erythrocytes. Synthetic peptides representing the predominant sequences of individual repeats were tested for their adhesive capacity for tick cell extract (TCE). Peptides containing acidic amino acids D or E at position 20 bound to TCE, while peptides with a G as the 20th amino acid were not adhesive to TCE. Antibodies produced in rabbits against a synthetic repeat peptide neutralized A. marginale infection of cultured tick cells, and the neutralization observed was similar to that effected by antibodies produced against the whole MSP1a recombinant protein. Analysis of tandemly repeated MSP1a peptides of several geographic isolates of A. marginale revealed a complex relationship between the msp1alpha genotype and the tick-transmissible phenotype of the isolate and suggested that both the sequence and conformation of the repeated peptides influenced the adhesive properties of MSP1a. These studies demonstrated that the tandemly repeated region of the protein mediates the adhesive function of MSP1a.
Vaccine, 2003
Identification of antigens that induce an immune response against tick infestations is required f... more Identification of antigens that induce an immune response against tick infestations is required for the development of vaccines against these economically important ectoparasites. In order to identify protective antigens, we constructed a cDNA expression library from a continuous Ixodes scapularis cell line (IDE8) that was initially derived from tick embryos. cDNA clones were subjected to several rounds of screening in which mice were immunized with individual pools and then challenge-exposed by allowing I. scapularis larvae to feed on the immunized and control mice. Immunity against tick infestation was determined by the reduction in the ability of the larvae to feed to repletion and molt to the nymphal stage. Individual clones in pools that induced immunity to larval infestations were partially sequenced and grouped according to their putative protein function by comparison with sequence databases. The screening identified several individual antigens that induced a protective immune response against I. scapularis infestations. Our studies demonstrated for the first time that cDNA expression library immunization (ELI) combined with sequence analysis is a powerful and efficient tool for identification of candidate antigens for use in vaccines against ticks.
Veterinary microbiology, Jan 14, 2009
Anaplasma marginale causes mild to severe hemoparasitic disease resulting in significant morbidit... more Anaplasma marginale causes mild to severe hemoparasitic disease resulting in significant morbidity and mortality in cattle worldwide. In the absence of universally efficacious vaccines, antimicrobial therapy combined with biocontainment and biosecurity strategies are critical to control anaplasmosis. Herein, we compared the effect of oxytetracycline, imidocarb and enrofloxacin on A. marginale isolates in short-term erythrocyte cultures. Electron micrographs detailing antimicrobial-induced changes in rickettsial morphology were scored (0-4) based on ultrastructural changes. These were compared to fluorochromatic changes detected by flow cytometry (FACS) using conversion of hydroethidine (HE) to ethidium bromide (EB) by living organisms to assess viability. A. marginale infectivity in selected cultures was confirmed by subinoculation into susceptible calves. Morphology scores were analyzed using Chi-squared tests and compared to FACS data by ANOVA with isolate, drug and concentration ...
Ticks and Tick-borne Diseases, 2014
Control of ticks on dogs is often done by application of repellents that contain permethrin as th... more Control of ticks on dogs is often done by application of repellents that contain permethrin as the active ingredient. In this research, we studied the role of a glutathione S-transferase (GST) gene in detoxification of permethrin by ticks using a gene silencing method RNA interference (RNAi). The brown dog tick, Rhipicephalus sanguineus, used in these studies, has a notable host preference for dogs, but also infests other mammals. In this research, R. sanguineus females were injected with gst double-stranded RNA (dsRNA) to effect gene silencing by RNAi and then exposed to sublethal doses of permethrin. Sixty hours after injection, the females were allowed to feed on sheep. The female ticks subjected to RNAi proved to be more susceptible to permethrin than the untreated controls. The effect of gene silencing was most notable in the highest dose group (50.3 ppm) in which all ticks died, while in the corresponding controls that were not subjected to RNAi this dose was not lethal. The acaricide treatment of the ticks resulted in a change in tick attachment behavior. Acaricide-treated ticks attached in a scattered pattern in contrast to the control ticks that attached and fed tightly clustered together. The time required for repletion for both the injected and non-injected females exposed to the higher permethrin level was shorter than that observed in the lower-dose groups and unexposed controls, and this more rapid attachment and feeding would likely favor more rapid transmission of pathogens. However, engorgement and egg mass weights were not significantly different among the experimental groups. This research demonstrated that the silencing of the gst gene increased the tick's susceptibility to permethrin. Overall, these results have contributed to our understanding of the detoxification mechanism of ticks and provide new considerations for the formulation of treatment strategies.
Veterinary Microbiology, 2009
Metarhizium anisopliae conidia were formulated in water or in olive oil containing 3% commercial ... more Metarhizium anisopliae conidia were formulated in water or in olive oil containing 3% commercial sunscreens (Everysun ® or E45 Sun Block 50 ® ) and exposed to an artiWcial UV source for up to 5 hours. Survival of conidia after 5 h of exposure to UV in oil formulation was 29% when protected with Everysun, 40% when protected with E45, and 4% in control. In comparison, survival of conidia formulated in water was 13% when protected with Everysun, 24% when protected with E45, and 0% in control. Furthermore, the inXuence of sunscreens on conidia viability and virulence to Rhipicephalus evertsi evertsi larvae and unfed adult ticks was evaluated. Adding these compounds to the conidial formulations did not reduce the viability of the conidia. Larval mortality was 95 and 100%, while unfed adult mortality was 90 and 97% after being exposed to unprotected conidia formulated in water or in oil, respectively. Conidia protected by Everysun or E45 formulated in water, induced 88 and 83% mortality in larvae, and 92 and 90% mortality in unfed adults, respectively. Conidia suspended in oil and protected by Everysun or E45 induced 94 and 91% mortality in larvae, and 83 and 81% in unfed adults, respectively. These observations indicate that olive oil and the two sunscreens confer protection to conidia against damages by UV radiation without interfering with their pathogenicity to ticks.
Vaccine, 2005
Antigens protective against Ixodes scapularis infestations were identified by cDNA expression lib... more Antigens protective against Ixodes scapularis infestations were identified by cDNA expression library immunization (ELI) and analysis of expressed sequenced tags (EST). Three cDNAs protective against larval tick infestations, 4F8, with homology to a nucleotidase, and 4D8 and 4E6 of unknown function, were characterized and obtained as recombinant proteins for immunization studies. Vaccination trials with recombinant proteins demonstrated an effect of these antigens against I. scapularis larvae in a mouse model. Herein, we evaluated the effect of recombinant antigens on I. scapularis adult infestations on immunized sheep. Vaccination with recombinant 4D8, 4F8, 4E6 and the combination of all three antigens reduced adult tick infestations by 58, 12, 20, and 16%, respectively, when compared to the control group but was statistically significant for 4D8 and 4F8 only. Oviposition was reduced by 22-49% in all groups immunized with recombinant tick antigens (P<0.05). The overall efficacy of vaccine formulations considering the effect on tick infestations and oviposition averaged 33-71%. These antigens, and especially 4D8, appear to be good candidates for continued development of a vaccine for control of tick infestations.
Comparative and Functional Genomics, 2009
The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes obligate tick-transmitted intracell... more The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes obligate tick-transmitted intracellular organisms, Anaplasma phagocytophilum and Anaplasma marginale that multiply in both vertebrate and tick host cells. Recently, we showed that A. marginale affects the expression of tick genes that are involved in tick survival and pathogen infection and multiplication. However, the gene expression profile in A. phagocytophilum-infected tick cells is currently poorly characterized. The objectives of this study were to characterize tick gene expression profile in Ixodes scapularis ticks and cultured ISE6 cells in response to infection with A. phagocypthilum and to compare tick gene expression responses in A. phagocytophilum- and A. marginale-infected tick cells by microarray and real-time RT-PCR analyses. The results of these studies demonstrated modulation of tick gene expression by A. phagocytophilum and provided evidence of different gene expression responses in tick cells infected with A. phagocytophilum and A. marginale. These differences in Anaplasma-tick interactions may reflect differences in pathogen life cycle in the tick cells.
Veterinary Microbiology, 2004
Infection of cells by tick-borne rickettsiae appears to be mediated by outer membrane proteins th... more Infection of cells by tick-borne rickettsiae appears to be mediated by outer membrane proteins that allow pathogens to adhere to host cells. Major surface protein (MSP) 1a of Anaplasma marginale, the type species for the genus Anaplasma, was shown previously to be an adhesin for tick cells. The A. marginale MSP1a has a variable number of tandem 28 or 29 amino acid repeats located in the amino terminal region of the protein that contains an adhesion domain that is necessary and sufficient for infection of tick cells. The MSP1a studies demonstrated the importance of combining structural and functional characteristics for identification of adhesive proteins. In the present study other outer membrane proteins containing tandem repeats were selected from organisms of the family Anaplasmataceae and studied for their adhesive properties to tick cells. The adhesive properties and protein characteristics were then analyzed in order to provide a predictor of the adhesion function of proteins identified from genome sequences. Proteins selected included the A. marginale MSP1a, A. phagocytophilum 100 and 130 kDa, Ehrlichia chaffeensis 120 kDa, E. canis 140 kDa and E. ruminantium "mucin", which were all cloned and expressed in Escherichia coli and then tested as adhesins for cultured IDE8 cells. Of the proteins studied, the A. marginale MSP1a and the E. ruminantium "mucin" were found to be adhesins for tick cells. Although all of these recombinant outer membrane proteins were glycosylated, the A. marginale MSP1a and E. ruminantium "mucin" adhesins shared a common feature of having a high Ser/Thr content in the tandem repeats. The results reported herein provide new information on the role of E. ruminantium "mucin" as an adhesin for tick cells and also suggest a role of glycans in adhesin molecules.
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Papers by Katherine Kocan