Expansion of zoonotic babesiosis and reported human cases, Connecticut, 2001-2010.

To document the expansion of human babesiosis in Connecticut, we analyzed reservoir host sera for seroreactivity to Babesia microti Franca and reviewed Connecticut human surveillance case data collected during 2001-2010. Sera from white-footed mice, Peromyscus leucopus Rafinesque, from 10 towns in 5 counties, collected at 4-7-yr periods between 2001 and 2010, were tested for total immunoglobulins. The prevalence of B. microti-positive mice was compared with confirmed and probable human case reports tabulated by the Connecticut Department of Public Health. The highest babesiosis and rodent seroprevalence rates were in New London County, where this protozoan disease was first documented in the state. However, human cases and reservoir host infection increased significantly from 2001-2005 to 2005-2010 and in other parts of the state. Clinicians should be aware that the disease is not confined to long-established endemic areas of the state.

Biology of ticks.

Ticks are among the most significant blood-sucking arthropods worldwide. They transmit various pathogens that can cause disease and death in people, domesticated animals, and wildlife. Ticks have several morphologic features and physiologic mechanisms that facilitate host selection, ingestion of vertebrate blood, mating, survival, and reproduction. Although the natural history of ticks varies considerably among species, these arthropods are well-adapted to survive in tropical, temperate, and even subarctic habitats. Key factors, including the reversion of agricultural lands to forests and a close association between people and ticks, have greatly increased the risk of tick bite and human disease.

Serum antibodies to West Nile virus in naturally exposed and vaccinated horses.

A polyvalent ELISA and plaque reduction neutralization tests (PRNTs) were used to measure serum antibodies to West Nile virus (WNV) in horses naturally exposed to or vaccinated against this flavivirus in Connecticut and New York State, USA. Relying on a PRNT as a 'gold standard', the main objective was to validate a modified ELISA containing a recombinant WNV envelope protein antigen. It was also important to assess specificity by testing sera from horses that had other, undiagnosed illnesses. Sera for the latter study were obtained from 43 privately owned horses during 1995-1996. Analyses by an ELISA and a PRNT confirmed the presence of WNV antibodies in 21 (91%) of 23 sera from naturally exposed horses and in 85% of the 20 vaccinated subjects; overall results for both study groups were highly concordant (91% agreement). Humoral responses of naturally exposed and immunized horses were similar. Both serological tests were useful in confirming past infections with WNV, but there was no evidence that horses with undiagnosed illnesses were exposed to WNV prior to a 1999 outbreak in Connecticut, USA.

Antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti in white-footed mice.

Serum samples were obtained from white-footed mice (Peromyscus leucopus) in tick-infested areas of Connecticut during the period 2001 through 2003 and analyzed for antibodies to Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. Emphasis was placed on the evaluations of highly specific recombinant VlsE or protein (p) 44 antigens of B. burgdorferi and A. phagocytophilum, respectively, in a newly developed enzyme-linked immunosorbent assay (ELISA) as well as testing sera with whole-cell antigens by conventional ELISA or indirect fluorescent antibody staining methods. Of the 414 mouse sera analyzed, 310 (75%) had antibodies to whole-cell B. burgdorferi, whereas 157 (38%) were positive to the VlsE antigen. The latter nearly equaled the overall antibody prevalence rate (37%) computed when sera were tested separately with the p44 antigen. Mice were exposed to these pathogens and B. microti (antibody prevalence = 25%) in extreme northern Connecticut as well as the southern coastal areas of the state, thus indicating further geographic expansion of these infections. Fifty-three (13%) sera from widely separated sites had antibodies to all three pathogens. With expression and immunological recognition of VlsE and p44 antigens in P. leucopus, separate incorporation of these fusion proteins in an ELISA was very helpful in confirming past or current infections and in identifying specific foci for B. burgdorferi and A. phagocytophilum.

Seroprevalence of antibodies against Borrelia burgdorferi and Anaplasma phagocytophilum in cats.

OBJECTIVE: To determine whether cats in the northeastern United States develop serum antibodies against antigens of Borrelia burgdorferi and Anaplasma phagocytophilum and whether coinfection with the 2 organisms occurs. SAMPLE POPULATION: Serum samples from 84 healthy cats and 9 cats with lameness, fever, anorexia, or fatigue. PROCEDURE: Serum antibodies against B. burgdorferi and A. phagocytophilum were measured with an ELISA incorporating a whole-cell preparation or purified recombinant antigens, by means of Western blot analysis, or indirect fluorescent antibody (IFA) staining. RESULTS: ELISA results indicated that 44 of 93 (47%) sera contained antibodies against > or = 3 B. burgdorferi antigens, whereas 43 (46%) were reactive to whole-cell B. burgdorferi. Serum reactivity to protein 35, VlsE, and outer surface proteins A and F was most common. Seropositivity to > or = 3 antigens occurred at the same rate (5/9) in the 9 ill cats as in the 84 healthy cats (46% [39/84]). Of 13 sera reactive to recombinant antigens, 9 were seropositive as measured by Western blot testing with whole-cell antigen. Seropositivity rates of 30% and 38% were detected for antibodies against A phagocytophilum via IFA and ELISA testing, respectively. Fifteen (16%) sera had antibodies against both pathogens. CONCLUSIONS AND CLINICAL RELEVANCE: Cats living in areas infested by Ixodes scapularis ticks are exposed to B. burgdorferi and A. phagocytophilum and, in some instances, may be coinfected. Most cats appeared healthy. An ELISA incorporating specific recombinant antigens may be used adjunctively with Western blot and other assays to confirm B. burgdorferi and A. phagocytophilum infection in cats.

Comparative reactivity of human sera to recombinant VlsE and other Borrelia burgdorferi antigens in class-specific enzyme-linked immunosorbent assays for Lyme borreliosis.

A comparative study of human sera was conducted to determine which purified preparations of 11 recombinant antigens of Borrelia burgdorferi sensu stricto were diagnostically most important in enzyme-linked immunosorbent assays (ELISAs). To assess sensitivity, 20 serum samples obtained 1-6 weeks after onset of illness from 20 persons who had physician-diagnosed erythema migrans (EM) were tested for IgM and IgG antibodies. In tests for IgM antibody, seropositivity of > or = 25% was recorded when ELISAs had separate preparations of protein (p) 37, p41-G, outer-surface protein (Osp) C, OspE, OspF or VlsE antigens. Sera reacted most frequently (80% positive) with VlsE antigen in analyses for IgG antibodies. When results of both class-specific assays were considered for VlsE, OspC or OspF, 90% of the EM cases were serologically confirmed. Results of specificity testing with a further 59 sera from persons who had syphilis, louse-borne relapsing fever, oral infections, rheumatoid arthritis or human granulocytic ehrlichiosis and 28 normal sera indicated no false positive reactions when VlsE antigen was used in tests for IgM antibody. One of the 11 louse-borne relapsing fever sera cross-reacted with VlsE antigen in tests for IgG antibodies. Minor cross-reactivity also occurred when p37, OspC, OspE or OspF antigens were used. Overall, VlsE was the most suitable antigen for laboratory diagnosis of Lyme borreliosis during the early weeks of B. burgdorferi infection because of its high sensitivity and specificity.

Reactivity of dog sera to whole-cell or recombinant antigens of Borrelia burgdorferi by ELISA and immunoblot analysis.

Enzyme-linked immunosorbent assays (ELISAs) with separate preparations of 10 purified recombinant antigens of Borrelia burgdorferi sensu stricto were used to test sera from 36 dogs not vaccinated with whole cells of this agent and from five dogs vaccinated with whole-cell B. burgdorferi bacteria. All dogs lived in tick-infested areas of Connecticut and south-eastern New York state, USA. The non-vaccinated dogs had limb or joint disorder, lameness and fever during the period 1984-1991 and had antibodies to B. burgdorferi, as determined by a polyvalent ELISA with whole-cell antigen. In re-analyses of sera for total immunoglobulins in ELISAs with recombinant antigens, reactions were most frequently recorded when outer-surface protein (Osp) F, protein (p)35, p37, p39 and p-41G (a flagellin component) were tested separately. Western immunoblots of a subset of 16 sera, positive by ELISA with whole-cell antigen and representing a range of antibody titres (640-40960), verified immune responses to these or other lysed whole-cell antigens. Sera from vaccinated dogs contained antibodies to OspA, OspB, p22, p37 and p41-G. Therefore, serological reactions to OspF, p35 and p39 were the most important indicators of natural exposure to B. burgdorferi. Serum reactivities to these recombinant antigens in ELISAs can be used to help identify possible natural infections of canine borreliosis in dogs not vaccinated with whole-cell B. burgdorferi and to provide information on the geographic distribution of this bacterium.

Antibodies to granulocytic ehrlichiae in cattle from Connecticut.

Enzyme-linked immunosorbent assays (ELISA) with a purified recombinant 44-kDa protein and indirect fluorescent antibody (IFA) staining methods incorporating whole-cell antigens of the human granulocytic ehrlichiosis (HGE) agent were used to detect antibodies to Ehrlichia phagocytophila genogroup organisms in cattle sera. The cattle lived in tick-infested areas of Connecticut, USA and were healthy at the times blood samples were collected in 1990, 1999 and 2000. Of the 339 serum samples analysed, 40 (12%) and 15 (4%) were positive by ELISA and IFA, respectively. Western immunoblots of a subset of sera verified antibody reactivity of six serum samples, positive by ELISA with titres of 640-2,560, to a protein with a molecular mass of c. 44 kDa. Although seroprevalence rates were low, cattle were exposed to the HGE agent at different sites and should be monitored for anaemia, leukopenia or thrombocytopenia, especially if there is evidence of unexplained decreased milk production. Different serological testing methods should be used to detect immunoglobulins.

A recombinant envelope protein-based enzyme-linked immunosorbent assay for West Nile virus serodiagnosis.

Recombinant West Nile virus envelope (E) protein was examined in enzyme-linked immunosorbent assay (ELISA) to detect antibodies elicited during West Nile virus infection. Horses (nine of 10) and humans (six of six) with confirmed West Nile virus infection had IgG and/or IgM antibodies to the E protein. Antibodies to the recombinant West Nile virus membrane and nonstructural 1 proteins were not detected in any of these sera. An E protein-based ELISA may aid in the serological diagnosis of West Nile virus infection.

Use of recombinant antigens of Borrelia burgdorferi and Anaplasma phagocytophilum in enzyme-linked immunosorbent assays to detect antibodies in white-tailed deer.

Serum samples obtained from white-tailed deer (Odocoileus virginianus) in Connecticut (n=218) and South Carolina (n=20) (USA) during the period 1992-2002 were analyzed for antibodies to whole-cell or recombinant antigens (i.e., fusion proteins) of Borrelia burgdorferi sensu stricto and Anaplasma phagocytophilum, etiologic agents of Lyme borreliosis and granulocytic ehrlichiosis, respectively. In enzyme-linked immunosorbent assays (ELISAs) with whole-cell B. burgdorferi, the overall seropositivity rate for Connecticut (53%) exceeded that for South Carolina (30%). In separate tests of seven recombinant antigens of B. burgdorferi by an ELISA, seroprevalence for the VlsE antigen was highest (48%) in Connecticut followed by outer surface protein (OspF) (21%), whereas serum reactivities to the protein (p) 41-G antigen (55%) and VlsE (25%) were most frequent for South Carolina sera. In analyses for antibodies to the recombinant protein (p) 44 antigen of A. phagocytophilum, seroprevalences of 52% and 25% were recorded for Connecticut and South Carolina samples, respectively. These findings paralleled those determined by indirect fluorescent antibody staining methods with whole cells (43% and 30%). Moreover, there was good agreement (74%) in results of Western blot analyses and an ELISA when a subset of 39 sera was screened with whole-cell or recombinant p44 antigens of A. phagocytophilum. An ELISA with highly specific recombinant VlsE or p44 antigens can be used in conjunction with other antibody tests to determine whether deer living in different regions of eastern United States were exposed to B. burgdorferi or A. phagocytophilum.

A comparison of serologic tests for the detection of serum antibodies to whole-cell and recombinant Borrelia burgdorferi antigens in cattle.

Serum samples from healthy dairy and beef cattle, living in tick-infested areas of Connecticut, USA, were analyzed by polyvalent enzyme-linked immunosorbent assays (ELISA), indirect fluorescent antibody (IFA) staining methods, or Western blot procedures to detect antibodies to tick-borne agents. Of the 80 sera tested by ELISA with whole-cell or 10 separate recombinant antigens (fusion proteins) of Borrelia burgdorferi sensu stricto, 57 (71%) were positive to 1 or more antigens, while 36 (45%) reacted to whole-cell antigens by IFA staining methods. Three (4%) of 80 samples had antibodies to Anaplasma phagocytophilum. There were antibodies to outer surface protein (Osp) A, OspB, OspC, OspE, OspF, protein (p) 41-G, p35, p37, and VlsE antigens of B. burgdorferi, but there was no reactivity to the p39 antigen by ELISA. Western immunoblots of a subset of 9 sera verified antibody presence in all samples and showed distinct reactivities to multiple proteins having molecular masses of about 31 kilodaltons (kDa), 34 kDa, 35 kDa, 41 kDa, and 83/93 kDa. High specificity (97%) was noted when 16 cattle sera containing antibodies to Leptospira interrogans serovars, Brucella sp., Anaplasma marginale, or A. phagocytophilum were tested by ELISA with separate whole-cell or recombinant B. burgdorferi antigens. An ELISA and Western blot analyses can be used to confirm the exposure of cattle to B. burgdorferi.

Serum antibodies to Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti in recaptured white-footed mice.

A mark-release-recapture study was conducted during 2007 through 2010 in six, tick-infested sites in Connecticut, United States to measure changes in antibody titers for Borrelia burgdorferi sensu stricto, Anaplasma phagocytophilum, and Babesia microti in Peromyscus leucopus (white-footed mice). There was an overall recapture rate of 40%, but only four tagged mice were caught in ≥2 yr. Sera from 561 mice were analyzed for total antibodies to B. burgdorferi and A. phagocytophilum by using whole-cell or recombinant (VlsE or protein 44) antigens in a solid-phase enzyme-linked immunosorbent assay or to whole-cell B. microti by indirect fluorescent antibody staining. Antibody prevalences were highly variable for B. burgdorferi (from 56% to 98%), A. phagocytophilum (from 11% to 85%), and B. microti (from 11% to 84%) depending on the site and time of sampling. Of 463 mice with antibodies, 206 (45%) had antibodies to all three pathogens. Changes in antibody status for some mice from negative to positive (117 seroconversions) or from positive to negative (55 reversions) were observed. Seroconversions were observed in 10.1% of 417 mice for B. burgdorferi, 18.0% of 306 mice for A. phagocytophilum, and 6.6% of 304 mice for B. microti; reversion rates were 5.3, 5.9, and 4.9%, respectively. Antibodies to all pathogens persisted in some mice over several weeks while, in others, there were marked declines in titration end points to negative status. The latter may indicate elimination of a certain pathogen, such as A. phagocytophilum, or that mouse immune systems ceased to produce antibodies despite an existing patent infection.

Seroprevalence of Powassan virus in New England deer, 1979-2010.

Powassan virus and its subtype, deer tick virus, are closely related tick-borne flaviviruses that circulate in North America. The incidence of human infection by these agents appears to have increased in recent years. To define exposure patterns among white-tailed deer, potentially useful sentinels that are frequently parasitized by ticks, we screened serum samples collected during 1979-2010 in Connecticut, Maine, and Vermont for neutralizing antibody by using a novel recombinant deer tick virus-West Nile virus chimeric virus. Evidence of exposure was detected in all three states. Overall our results demonstrate that seroprevalence is variable in time and space, suggesting that risk of exposure to Powassan virus is similarly variable.

Serum antibodies to whole-cell and recombinant antigens of Borrelia burgdorferi in cottontail rabbits.

Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985-86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37, or VlsE) during different seasons, but there was no reactivity to outer surface protein (Osp)A or OspB. Seventy-six of the 102 sera (75%) analyzed were reactive with one or more of the antigens; 61 of the positive samples (80%) reacted to whole-cell antigens, followed by results for the p35 (58%, 44/76), VlsE (43%, 33/76), and p37 (29%, 22/ 76) antigens. Fifty-eight sera (76%) contained antibodies to the VlsE or p35 antigens with or without reactivity to whole-cell antigens. High antibody titers (≥1:2,560) recorded for 52 sera indicate robust antibody production. In analyses for IgM antibodies in an ELISA containing whole-cell antigens, there were 30 positive sera; titers ranged from 1:160 to 1:640. There was minimal cross-reactivity when rabbit antisera to Treponema pallidum or four serovars of Leptospira interrogans were screened against B. burgdorferi antigens. Based on more-specific results, VlsE and p35 antigens appear to be useful markers for detecting possible B. burgdorferi infections.

Seasonal prevalence of serum antibodies to whole cell and recombinant antigens of Borrelia burgdorferi and Anaplasma phagocytophilum in white-tailed deer in Connecticut.

Whole-blood samples were obtained from 214 white-tailed deer (Odocoileus virginianus) representing 44 sites in Connecticut (USA) during 1992, 1993, 1996, 1999, and 2000 through 2006. Sera were analyzed for total antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto and Anaplasma phagocytophilum, the respective causative agents of Lyme borreliosis and human granulocytic anaplasmosis. Deer sera contained antibodies to both bacteria during different seasons and throughout the 11-yr study. Of the 224 sera tested, 141 (63%) contained antibodies to whole-cell B. burgdorferi in a polyvalent enzyme-linked immunosorbent assay, whereas 124 (55%) were positive to whole-cell A. phagocytophilum by indirect fluorescent antibody staining. Use of highly specific recombinant antigens (VlsE of B. burgdorferi and protein 44 of A. phagocytophilum) provided strong confirmatory results of past or current infections. There was coexistence of antibodies to whole-cell or recombinant antigens of both agents in 72 (32%) sera. Analyses of 18 sera from eight deer that were marked, released, and recaptured, showed minimal changes in antibody titer over sampling time intervals ranging from 17 days to 5.1 yr. Relatively high antibody prevalences for both bacterial agents in different seasons and years reaffirm that there are well-established foci for both tick-borne infections and probably reflect frequent exposure of deer to infected Ixodes scapularis ticks. November and December is a suitable period to obtain blood samples from deer to conduct serosurveillance for both bacteria.