Vol. 38, no. 2
Journal of Vector Ecology
401
Scientific Note Survey of adult Ixodes scapularis Say for disease agents in Mississippi Lauren Goltz1, Andrea Varela-Stokes2, and Jerome Goddard1 Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mississippi State, MS 39762, U.S.A., [email protected] 2 Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, U.S.A.
1
Ixodes scapularis, the black-legged tick, is a North American hard tick species capable of transmitting several pathogens of medical significance across a breadth of vertebrate hosts, including humans. It is now considered the most medically important tick in the United States, mainly due to its vector status for Borrelia burgdorferi, Babesia microti, and Anaplasma phagocytophilum, the causative agents of Lyme disease, babesiosis, and human granulocytic anaplasmosis, respectively (Burgdorfer et al. 1982, Gray et al. 2002, Dumler 2011, Telford et al. 2011). From the time it was first identified as a significant disease vector, scientists have evaluated the disease potential of I. scapularis in the northern and central U.S., primarily in relation to Lyme disease (LD) (Ostfeld et al. 1995, Piesman 2002, Diuk-Wasser et al. 2012), babesiosis (Spielman et al. 1981), and to a lesser extent, A. phagocytophilum (Adelson et al. 2004). However, little is known of the disease potential of I. scapularis in the southern U.S. where its range extends. A limited survey of ticks in Mississippi found I. scapularis infected with an unidentified spotted fever group rickettsial species but none infected with Borrelia organisms (Goddard et al. 2003). There has been at least one adult I. scapularis found infected in Mississippi with Anaplasma phagocytophilum (J. Goddard and A.S. Varela-Stokes, unpublished data), and to our knowledge no one has surveyed ticks in Mississippi for Babesia organisms. There have been numerous cases of babesiosis reported from the U.S., mostly due to Ba. microti in the northeastern U.S. (CDC 2012), however other novel or emerging Babesia species have been occasionally documented causing human illness in the U.S., such as Ba. duncani, Babesia sp. MO1, and Babesia sp. CA-1 (Persing et al. 1995, Herwaldt et al. 1996, Beattie et al. 2002, Herwaldt et al. 2004, Conrad et al. 2006). Babesiosis is currently not a reportable disease in Mississippi (CDC 2012) so we have no information about its occurrence in the state. Therefore, the objective of this study was to survey adult I. scapularis collected in northern Mississippi for molecular evidence of three different genera of disease agents: Borrelia, Babesia, and Anaplasma. Two 0.5 hectare sites in northern Mississippi were selected for tick sampling, one located at Wall Doxey State Park near Holly Springs in Marshall County and the other approximately 350 km southeast in Noxubee National Wildlife Refuge near Starkville (Oktibbeha County). These locations were chosen because they were wooded with a medium-dense canopy and contained leaf litter, and suitable host animals for I. scapularis. During a one-year period from
August 1, 2010 through July 31, 2011, each site was visited once per week, totaling 104 site visits. In order to collect from the plots in an efficient manner, each site was visually divided into 25 lanes and collecting was performed by transecting the sites in these predetermined lanes. A drag cloth was used for sampling and we checked for ticks every 10 m; all ticks found attached to the cloth were removed and placed in vials containing 95% ethanol. Ticks were identified to species and life stage, and adults were sexed. All immature Ixodes collected, as well as eight adults, were sent to Dr. Richard Robbins (Armed Forces Pest Management Board, Washington DC) for confirmation. A few specimens have been deposited as vouchers in the Mississippi Entomological Museum, Mississippi State University. Tick DNA was extracted according to manufacturer’s protocol using the Illustra tissue and cells genomic prep mini spin kit (GE Healthcare, Piscataway, NJ). Water controls were included with every set of samples subjected to PCR assay. To confirm successful extraction, all samples were tested by PCR for a portion of the tick mitochondrial 16S rRNA gene. For detection of A. phagocytophilum, the major surface protein gene, msp2, was amplified using msp2-3f and msp2-3r primers (Massung and Slater 2003). For detection of Babesia spp., testing was performed targeting the 18S rRNA gene using primers KIM18SF (5’-GAAATTAGAGTGTTTC-3’) and KIMREV2 (5’-ACCCTATTTAGCAGGTAAG-3’) (M. Yabsley, unpublished data). Tick extracts that demonstrated a product with initial PCR of the 18S rRNA target were subsequently re-tested using a nested PCR protocol to amplify another region of the 18S rRNA gene of apixomplexans using primers 5.1 and 3.1 in the primary amplification and RLBH-F and RLBH-R in the secondary amplification (Yabsley et al. 2005). To test for Borrelia spp., we used a nested PCR targeting the flaB gene using the primers FLALL and FLARL for primary amplification and FLARS and FLALS for secondary amplification (Barbour et al. 1996). Samples that produced amplicons in the flaB PCR assay were then subjected to a nested PCR targeting the 16S rRNA gene. The nested PCR used outer primers 16S1A and 16S1B in the primary amplification and inner primers 16S2A and 16S2B in the secondary amplification (Richter et al. 2003). Thermal cycler conditions were as described (Richter et al. 2003) with five additional cycles in both primary and secondary PCR amplifications and an annealing temperature adjusted to 62º C in the primary amplification. PCR products were visualized by electrophoresis in 2% agarose gels containing ethidium
402
Journal of Vector Ecology
bromide. Positive controls were included in all PCR assays. The positive control for tick mitochondrial 16S rRNA PCR assays was genomic DNA from a male Gulf Coast tick, Amblyomma maculatum. The positive control for A. phagocytophilum was genomic DNA from cultured A. phagocytophilum; for Babesia we used DNA from a clinical sample of infected whole dog blood obtained from Mississippi State College of Veterinary Medicine; and finally, genomic DNA extracted from cultured B. burgdorferi (Sh-2-82) was used as a control in PCR assays for Borrelia species. A total of 256 adult and six nymphal I. scapularis were collected in the sites over the one-year period. The paucity of nymphs collected reflects that in other studies and suggests that I. scapularis nymphs do not quest as high in vegetation in southern regions (Piesman 2002, Goddard and Piesman 2006). Of the 256 adults collected, 244 were examined for presence of disease agents (the six nymphs were not included due to their use in another, unrelated research project). Amplicons from amplification of the tick mitochondrial 16S rRNA gene were detected in 100% of tick samples, indicating that all DNA extractions were successful. No tick samples were positive for DNA of Anaplasma phagocytophilum by PCR of the msp2 gene. Initially, 82 samples were positive by nested PCR for Borrelia sp. when targeting the flaB gene, but upon subsequent PCR using the 16sRNA gene, they were all negative, indicating that the initial results were likely false positives due to laboratory contamination with flaB amplicons at that time. Twelve PCR products were amplified using the 18S rRNA primers (Yabsley et al. 2005) for Babesia. Upon sequencing, four of the ticks matched Ba. odocoilei, a non-zoonotic cervid babesiosis which is transmitted by I. scapularis (Waldrup et al. 1989a) and is prevalent through the southern United States, from eastern Texas (Waldrup et al. 1989b) through Florida (Telford and Forrester 1991), and southern Virginia (Perry et al. 1985). These four sequences have been submitted to GenBank. The remaining eight sequences had 99-100% identity to a fragment of the 18S rRNA of I. affinis. The significance of this finding is unclear since all adult I. scapularis collected in our study from late April through June (the period of activity of I. affinis) were sent to Dr. Rich Robbins (Armed Forces Pest Management Board, Washington, D.C.) for confirmation. In the southern U.S., I. scapularis infection rates with Borrelia burgdorferi are much lower (and hence, lower numbers of LD cases) (Goddard et al. 2012). This is often attributed to the fact that immature stages of I. scapularis feed primarily on lizards, which may be incompetent as reservoirs and incapable of infecting ticks (Piesman 2002). Another factor affecting the dynamics of LD is the fact that nymphal I. scapularis are the stage primarily biting people and transmitting the disease agent in the Northeast, whereas in the South, nymphal I. scapularis rarely, if ever, bite humans (Goddard 2002, Piesman 2002, Diuk-Wasser et al. 2006). Based on the relatively low number of I. scapularis collected in our study as well as our negative PCR results for human pathogens, I. scapularis appears to represent a low disease risk to humans in north Mississippi. This is supported by the
December 2013
previous survey of tick diseases in Mississippi (Goddard et al. 2003). These data support the idea that there is little evidence that I. scapularis is involved in a significant amount of disease transmission in Mississippi at this time. Acknowledgments Whitney Smith (Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University) provided technical assistance with this project. This article has been approved for publication as Journal Article No. J-12199 of the Mississippi Agriculture and Forestry Experiment Station, Mississippi State University. REFERENCES CITED Adelson, M.E., R.V. Rao, R.C. Tilton, K. Cabets, E. Eskow, L. Fein, J.L. Occi, and E. Mordechai. 2004. Prevalence of Borrelia burgdorferi, Bartonella spp., Babesia microti, and Anaplasma phagocytophilum in Ixodes scapularis ticks collected in northern New Jersey. J. Clin. Microbiol. 42: 2799-2801. Barbour, A.G., G.O. Maupin, G.J. Teltow, C.J. Carter, and J. Piesman. 1996. Identification of an uncultivable Borrelia species in the hard tick Amblyomma americanum: Possible agent of a Lyme disease-like illness. J. Infect. Dis. 173: 403-409. Beattie, J.F., M.L. Michelson, and P.J. Holman. 2002. Acute babesiosis caused by Babesia divergens in a resident of Kentucky. N. Engl. J. Med. 347: 697-698. Burgdorfer, W., A.G. Barbour, S.F. Hayes, J.L. Benach, E. Grunwaldt, and J. P. Davis. 1982. Lyme disease- a tickborne spirochetosis? Science 216: 1317-1319. CDC. 2012. Babesiosis surveillance -- 18 states, 2011. CDC, MMWR, 61: 505-509. Conrad, P.A., A.M. Kjemtrup, R.A. Carreno, J. Thomford, K. Wainwright, M. Eberhard, R. Quick, S.R. Telford, 3rd, and B.L. Herwaldt. 2006. Description of Babesia duncani n.sp. (Apicomplexa: Babesiidae) from humans and its differentiation from other piroplasms. Int. J. Parasitol. 36: 779-789. Diuk-Wasser, M.A., A.G. Gatewood, M.R. Cortinas, S. Yaremych-Hamer, J. Tsao, U. Kitron, G. Hickling, J.S. Brownstein, E. Walker, J. Piesman, and D. Fish. 2006. Spatiotemporal patterns of host-seeking Ixodes scapularis nymphs (Acari: Ixodidae) in the United States. J. Med. Entomol. 43: 166-176. Diuk-Wasser, M.A., A.G. Hoen, P. Cislo, R. Brinkerhoff, S.A. Hamer, M. Rowland, R. Cortinas, G. Vourc’h, F. Melton, G.J. Hickling, J.I. Tsao, J. Bunikis, A.G. Barbour, U. Kitron, J. Piesman, and D. Fish. 2012. Human risk of infection with Borrelia burgdorferi, the Lyme disease agent, in eastern United States. Am. J. Trop. Med. Hyg. 86: 320-327. Dumler, J.S. 2011. Ehrlichiosis and anaplasmosis, In: R.L. Guerrant, D.H. Walker, and P.F. Weller (eds.), Tropical Infectious Diseases: Principles, Pathogens, and Practice, 3rd ed. pp. 339-344. Saunders Elsevier, New York.
Vol. 38, no. 2
Journal of Vector Ecology
Goddard, J. 2002. A ten-year study of tick biting in Mississippi: implications for human disease transmission. J. Agromed. 8: 25-32. Goddard, J. and J. Piesman. 2006. New records of immature Ixodes scapularis from Mississippi. J. Vector Ecol. 31: 421-422. Goddard, J., J.W. Sumner, W.L. Nicholson, C.D. Paddock, J. Shen, and J. Piesman. 2003. Survey of ticks collected in Mississippi for Rickettsia, Ehrlichia, and Borrelia species. J. Vector Ecol. 28: 184-189. Goddard, J., A. Varela-Stokes, and R.W. Finley. 2012. Lymedisease-like illnesses in the South. J. Mississippi State Med. Assoc. 53: 68-72. Gray, J., O. Kahl, R.S. Lane, and G. Stanek. 2002. Lyme Borreliosis: Biology, Epidemiology, and Control. CABI Publishing, New York. Herwaldt, B., D.H. Persing, E.A. Precigout, W.L. Goff, D.A. Mathiesen, P.W. Taylor, M.L. Eberhard, and A.F. Gorenflot. 1996. A fatal case of babesiosis in Missouri: identification of another piroplasm that infects humans. Ann. Intern. Med. 124: 643-650. Herwaldt, B.L., G. de Bruyn, N.J. Pieniazek, M. Homer, K.H. Lofy, S.B. Slemenda, T.R. Fritsche, D.H. Persing, and A.P. Limaye. 2004. Babesia divergens-like infection, Washington State. Emerg. Infect. Dis. 10: 622-629. Massung, R.F. and K.G. Slater. 2003. Comparison of PCR assays for detection of the agent of human granulocytic ehrlichiosis. J. Clin. Microbiol. 41: 717-722. Ostfeld, R.S., O.M. Cepeda, K.R. Hazler, and M.C. Miller. 1995. Ecology of Lyme disease: habitat associations of ticks (Ixodes scapularis) in a rural landscape. Ecol. Appl. 5: 353-361. Perry, B.D., D.K. Nichols, and E.S. Cullom. 1985. Babesia odocoilei in white-tailed deer in Virginia. J. Wildl. Dis. 21: 149-152.
403
Persing, D.H., B.L. Herwaldt, C. Glaser, R.S. Lane, J.W. Thomford, D. Mathiesen, P.J. Krause, D.F. Phillip, and P.A. Conrad. 1995. Infection with a Babesia-like organism in northern California. N. Engl. J. Med. 332: 298-303. Piesman, J.F. 2002. Ecology of Borrelia burgdorferi sensu lato in North America. In: J.S. Gray, O. Kahl, R.S. Lane, and G. Stanek (eds.), Lyme Borreliosis -- Biology, Epidemiology, and Control. pp. 223-249. CABI Publishing, New York. Richter, D., D.B. Schlee, and F.-R. Matuschka. 2003. Relapsing fever-like spirochetes infecting European vector tick of Lyme Disease agent. Emerg. Infec. Dis. 9: 697-701. Spielman, A., P. Etkind, J.F. Piesman, T.K. Ruebush, 2nd., D.D. Juranek, and M.S. Jacobs. 1981. Reservoir hosts of human babesiosis on Nantucket Island. Am. J. Trop. Med. Hyg. 30: 560-565. Telford, S.R., 3rd, and D.J. Forrester. 1991. Piroplasms of white-tailed deer in Florida. Florida Field Nat. 19: 49-51. Telford, S.R. I., P.F. Weller, and J.H. Maquire. 2011. Babesiosis. pp. 676-681. In: R.L. Guerrant, D.H. Walker, and P.F. Weller (eds.), Tropical Infectious Diseases: Principles, Pathogens, and Practice, 3rd ed. Saunders Elsevier, New York. Varela, A.S., V.A. Moore, and S.E. Little. 2004. Disease agents in Amblyomma americanum from northeastern Georgia. J. Med. Entomol. 41: 753-759. Waldrup, K.A., A.A. Kocan, R.W. Barker, and G.G. Wagner. 1989a. Transmission of Babesia odocoilei in white-tailed deer (Odocoileus virginianus) by Ixodes scapularis (Acari: Ixodidae). J. Wildl. Dis. 26: 390-391. Waldrup, K.A., A.A. Kocan, T.Q. Qureshi, D.S. Davis, D. Baggett, and G.G. Wagner. 1989b. Serological prevalence and isolation of Babesia odocoilei in white-tailed deer in Texas and Oklahoma. J. Wildl. Dis. 25: 194-201. Yabsley, M.J., W.R. Davidson, D.E. Stallknecht, A. Varela, P.K. Swift, J.C. Devos, and S.A. Dubay. 2005. Evidence of tickborne organisms in mule deer from the western United States. Vector Borne Zoonot. Dis 5: 351-362.
Journal of Vector Ecology
401
Scientific Note Survey of adult Ixodes scapularis Say for disease agents in Mississippi Lauren Goltz1, Andrea Varela-Stokes2, and Jerome Goddard1 Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mississippi State, MS 39762, U.S.A., [email protected] 2 Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, U.S.A.
1
Ixodes scapularis, the black-legged tick, is a North American hard tick species capable of transmitting several pathogens of medical significance across a breadth of vertebrate hosts, including humans. It is now considered the most medically important tick in the United States, mainly due to its vector status for Borrelia burgdorferi, Babesia microti, and Anaplasma phagocytophilum, the causative agents of Lyme disease, babesiosis, and human granulocytic anaplasmosis, respectively (Burgdorfer et al. 1982, Gray et al. 2002, Dumler 2011, Telford et al. 2011). From the time it was first identified as a significant disease vector, scientists have evaluated the disease potential of I. scapularis in the northern and central U.S., primarily in relation to Lyme disease (LD) (Ostfeld et al. 1995, Piesman 2002, Diuk-Wasser et al. 2012), babesiosis (Spielman et al. 1981), and to a lesser extent, A. phagocytophilum (Adelson et al. 2004). However, little is known of the disease potential of I. scapularis in the southern U.S. where its range extends. A limited survey of ticks in Mississippi found I. scapularis infected with an unidentified spotted fever group rickettsial species but none infected with Borrelia organisms (Goddard et al. 2003). There has been at least one adult I. scapularis found infected in Mississippi with Anaplasma phagocytophilum (J. Goddard and A.S. Varela-Stokes, unpublished data), and to our knowledge no one has surveyed ticks in Mississippi for Babesia organisms. There have been numerous cases of babesiosis reported from the U.S., mostly due to Ba. microti in the northeastern U.S. (CDC 2012), however other novel or emerging Babesia species have been occasionally documented causing human illness in the U.S., such as Ba. duncani, Babesia sp. MO1, and Babesia sp. CA-1 (Persing et al. 1995, Herwaldt et al. 1996, Beattie et al. 2002, Herwaldt et al. 2004, Conrad et al. 2006). Babesiosis is currently not a reportable disease in Mississippi (CDC 2012) so we have no information about its occurrence in the state. Therefore, the objective of this study was to survey adult I. scapularis collected in northern Mississippi for molecular evidence of three different genera of disease agents: Borrelia, Babesia, and Anaplasma. Two 0.5 hectare sites in northern Mississippi were selected for tick sampling, one located at Wall Doxey State Park near Holly Springs in Marshall County and the other approximately 350 km southeast in Noxubee National Wildlife Refuge near Starkville (Oktibbeha County). These locations were chosen because they were wooded with a medium-dense canopy and contained leaf litter, and suitable host animals for I. scapularis. During a one-year period from
August 1, 2010 through July 31, 2011, each site was visited once per week, totaling 104 site visits. In order to collect from the plots in an efficient manner, each site was visually divided into 25 lanes and collecting was performed by transecting the sites in these predetermined lanes. A drag cloth was used for sampling and we checked for ticks every 10 m; all ticks found attached to the cloth were removed and placed in vials containing 95% ethanol. Ticks were identified to species and life stage, and adults were sexed. All immature Ixodes collected, as well as eight adults, were sent to Dr. Richard Robbins (Armed Forces Pest Management Board, Washington DC) for confirmation. A few specimens have been deposited as vouchers in the Mississippi Entomological Museum, Mississippi State University. Tick DNA was extracted according to manufacturer’s protocol using the Illustra tissue and cells genomic prep mini spin kit (GE Healthcare, Piscataway, NJ). Water controls were included with every set of samples subjected to PCR assay. To confirm successful extraction, all samples were tested by PCR for a portion of the tick mitochondrial 16S rRNA gene. For detection of A. phagocytophilum, the major surface protein gene, msp2, was amplified using msp2-3f and msp2-3r primers (Massung and Slater 2003). For detection of Babesia spp., testing was performed targeting the 18S rRNA gene using primers KIM18SF (5’-GAAATTAGAGTGTTTC-3’) and KIMREV2 (5’-ACCCTATTTAGCAGGTAAG-3’) (M. Yabsley, unpublished data). Tick extracts that demonstrated a product with initial PCR of the 18S rRNA target were subsequently re-tested using a nested PCR protocol to amplify another region of the 18S rRNA gene of apixomplexans using primers 5.1 and 3.1 in the primary amplification and RLBH-F and RLBH-R in the secondary amplification (Yabsley et al. 2005). To test for Borrelia spp., we used a nested PCR targeting the flaB gene using the primers FLALL and FLARL for primary amplification and FLARS and FLALS for secondary amplification (Barbour et al. 1996). Samples that produced amplicons in the flaB PCR assay were then subjected to a nested PCR targeting the 16S rRNA gene. The nested PCR used outer primers 16S1A and 16S1B in the primary amplification and inner primers 16S2A and 16S2B in the secondary amplification (Richter et al. 2003). Thermal cycler conditions were as described (Richter et al. 2003) with five additional cycles in both primary and secondary PCR amplifications and an annealing temperature adjusted to 62º C in the primary amplification. PCR products were visualized by electrophoresis in 2% agarose gels containing ethidium
402
Journal of Vector Ecology
bromide. Positive controls were included in all PCR assays. The positive control for tick mitochondrial 16S rRNA PCR assays was genomic DNA from a male Gulf Coast tick, Amblyomma maculatum. The positive control for A. phagocytophilum was genomic DNA from cultured A. phagocytophilum; for Babesia we used DNA from a clinical sample of infected whole dog blood obtained from Mississippi State College of Veterinary Medicine; and finally, genomic DNA extracted from cultured B. burgdorferi (Sh-2-82) was used as a control in PCR assays for Borrelia species. A total of 256 adult and six nymphal I. scapularis were collected in the sites over the one-year period. The paucity of nymphs collected reflects that in other studies and suggests that I. scapularis nymphs do not quest as high in vegetation in southern regions (Piesman 2002, Goddard and Piesman 2006). Of the 256 adults collected, 244 were examined for presence of disease agents (the six nymphs were not included due to their use in another, unrelated research project). Amplicons from amplification of the tick mitochondrial 16S rRNA gene were detected in 100% of tick samples, indicating that all DNA extractions were successful. No tick samples were positive for DNA of Anaplasma phagocytophilum by PCR of the msp2 gene. Initially, 82 samples were positive by nested PCR for Borrelia sp. when targeting the flaB gene, but upon subsequent PCR using the 16sRNA gene, they were all negative, indicating that the initial results were likely false positives due to laboratory contamination with flaB amplicons at that time. Twelve PCR products were amplified using the 18S rRNA primers (Yabsley et al. 2005) for Babesia. Upon sequencing, four of the ticks matched Ba. odocoilei, a non-zoonotic cervid babesiosis which is transmitted by I. scapularis (Waldrup et al. 1989a) and is prevalent through the southern United States, from eastern Texas (Waldrup et al. 1989b) through Florida (Telford and Forrester 1991), and southern Virginia (Perry et al. 1985). These four sequences have been submitted to GenBank. The remaining eight sequences had 99-100% identity to a fragment of the 18S rRNA of I. affinis. The significance of this finding is unclear since all adult I. scapularis collected in our study from late April through June (the period of activity of I. affinis) were sent to Dr. Rich Robbins (Armed Forces Pest Management Board, Washington, D.C.) for confirmation. In the southern U.S., I. scapularis infection rates with Borrelia burgdorferi are much lower (and hence, lower numbers of LD cases) (Goddard et al. 2012). This is often attributed to the fact that immature stages of I. scapularis feed primarily on lizards, which may be incompetent as reservoirs and incapable of infecting ticks (Piesman 2002). Another factor affecting the dynamics of LD is the fact that nymphal I. scapularis are the stage primarily biting people and transmitting the disease agent in the Northeast, whereas in the South, nymphal I. scapularis rarely, if ever, bite humans (Goddard 2002, Piesman 2002, Diuk-Wasser et al. 2006). Based on the relatively low number of I. scapularis collected in our study as well as our negative PCR results for human pathogens, I. scapularis appears to represent a low disease risk to humans in north Mississippi. This is supported by the
December 2013
previous survey of tick diseases in Mississippi (Goddard et al. 2003). These data support the idea that there is little evidence that I. scapularis is involved in a significant amount of disease transmission in Mississippi at this time. Acknowledgments Whitney Smith (Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University) provided technical assistance with this project. This article has been approved for publication as Journal Article No. J-12199 of the Mississippi Agriculture and Forestry Experiment Station, Mississippi State University. REFERENCES CITED Adelson, M.E., R.V. Rao, R.C. Tilton, K. Cabets, E. Eskow, L. Fein, J.L. Occi, and E. Mordechai. 2004. Prevalence of Borrelia burgdorferi, Bartonella spp., Babesia microti, and Anaplasma phagocytophilum in Ixodes scapularis ticks collected in northern New Jersey. J. Clin. Microbiol. 42: 2799-2801. Barbour, A.G., G.O. Maupin, G.J. Teltow, C.J. Carter, and J. Piesman. 1996. Identification of an uncultivable Borrelia species in the hard tick Amblyomma americanum: Possible agent of a Lyme disease-like illness. J. Infect. Dis. 173: 403-409. Beattie, J.F., M.L. Michelson, and P.J. Holman. 2002. Acute babesiosis caused by Babesia divergens in a resident of Kentucky. N. Engl. J. Med. 347: 697-698. Burgdorfer, W., A.G. Barbour, S.F. Hayes, J.L. Benach, E. Grunwaldt, and J. P. Davis. 1982. Lyme disease- a tickborne spirochetosis? Science 216: 1317-1319. CDC. 2012. Babesiosis surveillance -- 18 states, 2011. CDC, MMWR, 61: 505-509. Conrad, P.A., A.M. Kjemtrup, R.A. Carreno, J. Thomford, K. Wainwright, M. Eberhard, R. Quick, S.R. Telford, 3rd, and B.L. Herwaldt. 2006. Description of Babesia duncani n.sp. (Apicomplexa: Babesiidae) from humans and its differentiation from other piroplasms. Int. J. Parasitol. 36: 779-789. Diuk-Wasser, M.A., A.G. Gatewood, M.R. Cortinas, S. Yaremych-Hamer, J. Tsao, U. Kitron, G. Hickling, J.S. Brownstein, E. Walker, J. Piesman, and D. Fish. 2006. Spatiotemporal patterns of host-seeking Ixodes scapularis nymphs (Acari: Ixodidae) in the United States. J. Med. Entomol. 43: 166-176. Diuk-Wasser, M.A., A.G. Hoen, P. Cislo, R. Brinkerhoff, S.A. Hamer, M. Rowland, R. Cortinas, G. Vourc’h, F. Melton, G.J. Hickling, J.I. Tsao, J. Bunikis, A.G. Barbour, U. Kitron, J. Piesman, and D. Fish. 2012. Human risk of infection with Borrelia burgdorferi, the Lyme disease agent, in eastern United States. Am. J. Trop. Med. Hyg. 86: 320-327. Dumler, J.S. 2011. Ehrlichiosis and anaplasmosis, In: R.L. Guerrant, D.H. Walker, and P.F. Weller (eds.), Tropical Infectious Diseases: Principles, Pathogens, and Practice, 3rd ed. pp. 339-344. Saunders Elsevier, New York.
Vol. 38, no. 2
Journal of Vector Ecology
Goddard, J. 2002. A ten-year study of tick biting in Mississippi: implications for human disease transmission. J. Agromed. 8: 25-32. Goddard, J. and J. Piesman. 2006. New records of immature Ixodes scapularis from Mississippi. J. Vector Ecol. 31: 421-422. Goddard, J., J.W. Sumner, W.L. Nicholson, C.D. Paddock, J. Shen, and J. Piesman. 2003. Survey of ticks collected in Mississippi for Rickettsia, Ehrlichia, and Borrelia species. J. Vector Ecol. 28: 184-189. Goddard, J., A. Varela-Stokes, and R.W. Finley. 2012. Lymedisease-like illnesses in the South. J. Mississippi State Med. Assoc. 53: 68-72. Gray, J., O. Kahl, R.S. Lane, and G. Stanek. 2002. Lyme Borreliosis: Biology, Epidemiology, and Control. CABI Publishing, New York. Herwaldt, B., D.H. Persing, E.A. Precigout, W.L. Goff, D.A. Mathiesen, P.W. Taylor, M.L. Eberhard, and A.F. Gorenflot. 1996. A fatal case of babesiosis in Missouri: identification of another piroplasm that infects humans. Ann. Intern. Med. 124: 643-650. Herwaldt, B.L., G. de Bruyn, N.J. Pieniazek, M. Homer, K.H. Lofy, S.B. Slemenda, T.R. Fritsche, D.H. Persing, and A.P. Limaye. 2004. Babesia divergens-like infection, Washington State. Emerg. Infect. Dis. 10: 622-629. Massung, R.F. and K.G. Slater. 2003. Comparison of PCR assays for detection of the agent of human granulocytic ehrlichiosis. J. Clin. Microbiol. 41: 717-722. Ostfeld, R.S., O.M. Cepeda, K.R. Hazler, and M.C. Miller. 1995. Ecology of Lyme disease: habitat associations of ticks (Ixodes scapularis) in a rural landscape. Ecol. Appl. 5: 353-361. Perry, B.D., D.K. Nichols, and E.S. Cullom. 1985. Babesia odocoilei in white-tailed deer in Virginia. J. Wildl. Dis. 21: 149-152.
403
Persing, D.H., B.L. Herwaldt, C. Glaser, R.S. Lane, J.W. Thomford, D. Mathiesen, P.J. Krause, D.F. Phillip, and P.A. Conrad. 1995. Infection with a Babesia-like organism in northern California. N. Engl. J. Med. 332: 298-303. Piesman, J.F. 2002. Ecology of Borrelia burgdorferi sensu lato in North America. In: J.S. Gray, O. Kahl, R.S. Lane, and G. Stanek (eds.), Lyme Borreliosis -- Biology, Epidemiology, and Control. pp. 223-249. CABI Publishing, New York. Richter, D., D.B. Schlee, and F.-R. Matuschka. 2003. Relapsing fever-like spirochetes infecting European vector tick of Lyme Disease agent. Emerg. Infec. Dis. 9: 697-701. Spielman, A., P. Etkind, J.F. Piesman, T.K. Ruebush, 2nd., D.D. Juranek, and M.S. Jacobs. 1981. Reservoir hosts of human babesiosis on Nantucket Island. Am. J. Trop. Med. Hyg. 30: 560-565. Telford, S.R., 3rd, and D.J. Forrester. 1991. Piroplasms of white-tailed deer in Florida. Florida Field Nat. 19: 49-51. Telford, S.R. I., P.F. Weller, and J.H. Maquire. 2011. Babesiosis. pp. 676-681. In: R.L. Guerrant, D.H. Walker, and P.F. Weller (eds.), Tropical Infectious Diseases: Principles, Pathogens, and Practice, 3rd ed. Saunders Elsevier, New York. Varela, A.S., V.A. Moore, and S.E. Little. 2004. Disease agents in Amblyomma americanum from northeastern Georgia. J. Med. Entomol. 41: 753-759. Waldrup, K.A., A.A. Kocan, R.W. Barker, and G.G. Wagner. 1989a. Transmission of Babesia odocoilei in white-tailed deer (Odocoileus virginianus) by Ixodes scapularis (Acari: Ixodidae). J. Wildl. Dis. 26: 390-391. Waldrup, K.A., A.A. Kocan, T.Q. Qureshi, D.S. Davis, D. Baggett, and G.G. Wagner. 1989b. Serological prevalence and isolation of Babesia odocoilei in white-tailed deer in Texas and Oklahoma. J. Wildl. Dis. 25: 194-201. Yabsley, M.J., W.R. Davidson, D.E. Stallknecht, A. Varela, P.K. Swift, J.C. Devos, and S.A. Dubay. 2005. Evidence of tickborne organisms in mule deer from the western United States. Vector Borne Zoonot. Dis 5: 351-362.
