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Antibody response in white-footed mice (Peromyscus leucopus) experimentally infected with the Lyme disease spirochete (Borrelia burgdorferi).

White-footed mice (Peromyscus leucopus), the primary reservoir for Borrelia burgdorferi in the northern midwest and northeastern United States, were experimentally inoculated with an infectious strain or a noninfectious strain of the Lyme disease spirochete and examined for their specific antibody response with the enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis. Immunoglobulin M (IgM) anti-B. burgdorferi antibodies were detected in mice 1 to 2 days after inoculation with either the infectious or noninfectious strain of spirochetes and peaked on days 4 and 5. Mice inoculated with the infectious strain of spirochete had a secondary increase in IgM 21 days after inoculation. Mice also produced both IgG1 and IgG2 antibodies beginning 5 to 7 days after inoculation and they increased in titer until 84 days after inoculation when the experiment was terminated. Western blot analysis of sequential plasma samples from mice inoculated with the infectious strain of spirochete demonstrated the development of IgM, IgG1, and IgG2 antibodies to numerous spirochetal antigens, whereas mice inoculated with the noninfectious strain had reduced blot patterns with antibodies reactive primarily to the 31,000-kilodalton outer surface protein A. Persistent spirochetal infection in some mice, in spite of a strong and diverse antibody response, deserves further investigation.

Animals↗

Peromyscus leucopus and Microtus pennsylvanicus simultaneously infected with Borrelia burgdorferi and Babesia microti.

Borrelia burgdorferi, the etiologic agent of Lyme disease, and Babesia microti, the causative agent of human babesiosis, were isolated from 71 and 57%, respectively, of 14 specimens of Peromyscus leucopus and Microtus pennsylvanicus collected from Prudence and Patience Islands, R.I. Both pathogens were isolated from five individual rodents. The presence of these two infectious organisms in the same mammal suggests that individual larval Ixodes dammini may ingest both pathogens and subsequently transmit them in the nymphal stage.

Animals↗

Seasonal prevalence of Borrelia burgdorferi in natural populations of white-footed mice, Peromyscus leucopus.

Borrelia burgdorferi, the etiologic agent of Lyme disease, was isolated from 111 of 237 Peromyscus leucopus captured during all seasons of the year. Borreliae were cultured from tissues of the spleen (101 mice), left kidney (76 mice), and right kidney (73 mice), from blood (12 mice), and from one fetus. Mice were infected during the winter, when immature Ixodes dammini were inactive. The prevalence of infection during the winter (less than or equal to 33%) was more than twofold lower than that during the summer (ca. 75%), a time when nymphal ticks are abundant. Overwintering, infected mice are reservoir hosts for subadult ticks that begin feeding in early spring. Twenty white-footed mice from which B. burgdorferi was isolated from tissues of spleen or kidney but not from blood were parasitized by larval I. dammini or Dermacentor variabilis which harbored borreliae. We conclude that these mice were infectious to feeding ticks, even though borreliae were not isolated from blood.

Animals↗

The urinary bladder, a consistent source of Borrelia burgdorferi in experimentally infected white-footed mice (Peromyscus leucopus).

White-footed mice, Peromyscus leucopus, were experimentally infected in the laboratory with Borrelia burgdorferi, the causative agent of Lyme disease. After mice were infected by intraperitoneal or subcutaneous inoculation or by tick bite, attempts were made to culture spirochetes from the urinary bladder, spleen, kidney, blood, and urine. Spirochetes were most frequently isolated from the bladder (94%), followed by the kidney (75%), spleen (61%), and blood (13%). No spirochetes were isolated from the urine. Tissue sectioning and immunofluorescence staining of the urinary bladder demonstrated spirochetes within the bladder wall. The results demonstrate that cultivation of the urinary bladder is very effective at isolating B. burgdorferi from experimentally infected white-footed mice and that culturing this organ may be productive when surveying wild rodents for infection with this spirochete.

Animals↗

Serologic analyses of Peromyscus leucopus, a rodent reservoir for Borrelia burgdorferi, in northeastern United States.

An enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent-antibody test were used to detect antibodies to Borrelia burgdorferi, the causative agent of Lyme disease, in Peromyscus leucopus (white-footed mouse). Of the 661 mice captured in Connecticut, Rhode Island, and New York during 1980 and 1983 to 1987, 166 (25.1%) had antibodies to B. burgdorferi by ELISA. Comparative analyses of 210 serum specimens, collected in areas where Lyme disease is endemic, revealed a threefold difference in sensitivity between the ELISA (38.1% positive) and the indirect fluorescent-antibody method (12.4%). Although prevalence of seropositive P. leucopus was highest during June, elevated amounts of antibody (1:1,280 to 1:2,560) were detected in mice that harbored spirochetes during all seasons. Being reservoirs for B. burgdorferi, these rodents are suitable for monitoring spirochete infections at foci and should be included in field evaluations of control programs aimed at suppressing Lyme disease.

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Efficacy of the urinary bladder for isolation of Borrelia burgdorferi from naturally infected, wild Peromyscus leucopus.

The efficacy of culturing urinary bladder tissue for Borrelia burgdorferi from naturally infected, wild Peromyscus leucopus mice was determined. The urinary bladder cultures were as efficient as spleen, kidney, and blood tissue cultures. The rapid B. burgdorferi isolation (mean, 6 days) from mouse urinary bladders should aid in defining new Lyme disease foci.

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Comparison of polymerase chain reaction and culture for detection of Borrelia burgdorferi in naturally infected Peromyscus leucopus and experimentally infected C.B-17 scid/scid mice.

Culture and the polymerase chain reaction (PCR) were compared for detection of Borrelia burgdorferi infection in wild-caught Peromyscus leucopus and experimentally inoculated C.B-17 scid/scid (severe combined immunodeficient) mice. PCR targeted highly conserved regions of the ospA gene and could detect one to five cultured organisms and 10 to 50 copies of molecularly cloned ospA DNA. Organs (kidney, spleen, and urinary bladder) and/or ear biopsy samples were obtained from 108 captured P. leucopus mice, and tissues were obtained from 7 experimentally inoculated mice. A simple sample-processing procedure with proteinase K and detergent treatment was used in the PCR analysis. Overall, B. burgdorferi was detected in 29 of 108 (27%) P. leucopus mice by culture and in 31 of 108 (29%) mice by PCR. As assessed by the kappa statistic, agreement between PCR and culture was high for ear and bladder (kappa = 0.80 and 0.65, respectively) and low for kidney and spleen (kappa = 0.37 and 0.03, respectively). While concordant results were obtained from 98 animals, PCR detected B. burgdorferi from 6 additional mice for which cultures were negative and culture detected B. burgdorferi from 4 animals which were PCR negative. Further phenol-chloroform extraction of DNA in a limited number of samples improved the sensitivity of PCR compared with that of culture. These results indicate that PCR may be as sensitive as culture for detecting B. burgdorferi in ear samples and that PCR analysis is suitable for establishing the infection status of animals in mark-release-recapture studies.

Animals↗

Detection of Borrelia burgdorferi in urine of Peromyscus leucopus by inhibition enzyme-linked immunosorbent assay.

An inhibition enzyme-linked immunosorbent assay was developed to detect Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, in urine from white-footed mice (Peromyscus leucopus). Of the 87 urine specimens tested from 87 mice collected in widely separated tick-infested sites in Connecticut, 57 (65.5%) contained detectable concentrations of spirochetal antigens. Forty-seven (62.7%) of 75 serum samples analyzed contained antibodies to B. burgdorferi. In culture work with tissues from bladders, kidneys, spleens, or ears, 50 of 87 mice (57.5%) were infected with B. burgdorferi. Thirty-eight (76%) of 50 infected mice had antigens of this spirochete in urine, while 36 (72%) individuals had infected bladders. Of those with infected bladders, 24 (66.7%) mice excreted subunits or whole cells of B. burgdorferi into urine. Successful culturing of B. burgdorferi from mouse tissues, the presence of serum antibodies to this bacterium, and detection of antigens to this spirochete in urine provide further evidence that multiple assays can be performed to verify the presence of B. burgdorferi in P. leucopus.

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Infection with agents of human granulocytic ehrlichiosis, lyme disease, and babesiosis in wild white-footed mice (Peromyscus leucopus) in Connecticut.

White-footed mice, Peromyscus leucopus, were captured in southern Connecticut during 1997 and 1998 to determine the prevalence of infections caused by granulocytic Ehrlichia sp., Borrelia burgdorferi, and Babesia microti. Of the 50 mice captured and recaptured, 25 of 47 (53.2%) and 23 of 48 (47.9%) contained antibodies to the BDS or NCH-1 Ehrlichia strains, respectively, as determined by indirect fluorescent antibody (IFA) staining methods. The majority (83.3%) of 48 mice also contained antibodies to B. burgdorferi, as determined by enzyme-linked immunosorbent assay. Moreover, 20 of 26 (76.9%) contained antibodies to B. microti by IFA staining methods. In nested PCR tests using the 16S rRNA gene, the DNA of the human granulocytic ehrlichiosis (HGE) agent was detected in 17 of 47 mice (36.2%), but only 4 (23.5%) of these 17 mice were PCR positive at each capture. Antibody-positive reactions to granulocytic Ehrlichia sp. organisms were detected in 17 of 23 (73. 9%) of the PCR-positive mice. The sequences from PCR products from nine positive blood samples were identical to the HGE agent. Ehrlichia spp. were cultured from three of five mice captured in April 1998, including one that was PCR positive in April 1997. In addition, 2 of 14 larval Ixodes scapularis pools, which were attached to two PCR-positive mice, contained DNA of the HGE agent. A high percentage of white-footed mice are infected or have been infected naturally by the HGE agent with low-level persistent infection or frequent reinfection in some individual mice. However, the changes noted in the presence of DNA and antibodies in repeated blood and serum samples from individual mice over several months of field collection suggests that infection with granulocytic Ehrlichia is transient in most wild P. leucopus.

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Isolation of the etiologic agent of human granulocytic ehrlichiosis from the white-footed mouse (Peromyscus leucopus).

We examined white-footed mice (Peromyscus leucopus) from Minnesota for infection with the etiologic agent of human granulocytic ehrlichiosis (HGE). From April to September 1997, we collected P. leucopus from Washington County, Minnesota, an area enzootic for HGE. Blood was cultivated in HL60 cells for isolation of the HGE agent. Of 59 mice examined, only a single mouse was culture positive for the HGE agent. The 16S ribosomal DNA sequence of the isolate was determined to be identical to that of the HGE agent. The isolate was reactive with monoclonal antibodies to the 44-kDa antigen of the HGE agent and was infectious for laboratory mice.

Animals↗

Molecular linkage of hantavirus pulmonary syndrome to the white-footed mouse, Peromyscus leucopus: genetic characterization of the M genome of New York virus.

The complete M segment sequences of hantaviruses amplified from tissues of a patient with hantavirus pulmonary syndrome in the northeastern United States and from white-footed mice, Peromyscus leucopus, from New York were 99% identical and differed from those of Four Corners virus by 23%. The serum of this patient failed to recognize a conserved, immunodominant epitope of the Four Corners virus G1 glycoprotein. Collectively, these findings indicate that P. leucopus harbors a genetically and antigenically distinct hantavirus that causes hantavirus pulmonary syndrome.

Amino Acid Sequence↗

Antibody responses to Four Corners hantavirus infections in the deer mouse (Peromyscus maniculatus): identification of an immunodominant region of the viral nucleocapsid protein.

Antibody responses to Four Corners hantavirus (FCV) infections in the deer mouse (Peromyscus maniculatus) were characterized by using FCV nucleocapsid protein (N), glycoprotein 1 (G1), and glycoprotein 2 (G2) recombinant polypeptides in Western immunoblot assays. Strong immunoglobulin G reactivities to FCV N were observed among FCV-infected wild P. maniculatus mice (n = 34) and in laboratory-infected P. maniculatus mice (n = 11). No immunoglobulin G antibody reactivities to FCV G1 or G2 linear determinants were detected. The strongest N responses were mapped to an amino-proximal segment between amino acids 17 and 59 (QLVTARQKLKDAERAVELDPDDVNKSTLQSRRAAVSALETKLG). FCV N antibodies cross-reacted with recombinant N proteins encoded by Puumala, Seoul, and Hantaan viruses.

Amino Acid Sequence↗

Sin Nombre virus pathogenesis in Peromyscus maniculatus.

Sin Nombre virus (SNV), a member of the Hantavirus genus, causes acute viral pneumonia in humans and is thought to persistently infect mice. The deer mouse, Peromyscus maniculatus, has been identified as the primary reservoir host for SNV. To understand SNV infection of P. maniculatus, we examined wild deer mice for localization of viral antigens and nucleic acid. Morphologic examination consistently revealed septal edema within lung tissue and mononuclear cell infiltrates in portal areas of the liver. Immunohistochemical analysis of SNV-infected deer mice identified viral antigens within lung, liver, kidney, and spleen. The lungs consistently presented with the highest levels of viral antigen by immunohistochemistry and with the highest levels of nucleic acid by reverse transcriptase (RT) PCR. The mononuclear cell infiltrates surrounding liver portal triads were positive for SNV antigens in addition to resident macrophages in liver sinuses. Spleen tissue contained antigens in both the red pulp and the periartereolar region of the white pulp. The kidney presented with no gross pathology, although antigens could be localized to glomeruli. Virus antigen levels within the kidney were highest in deer mice that did not have antibodies to SNV but contained viral nucleic acid detectable by RT PCR. Since transmission is thought to occur via urine, our results suggest that virus transmission may be highest in the early stages of infection. In addition, these results indicate that SNV does cause some pathology within its reservoir host.

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Role of maternal antibody in natural infection of Peromyscus maniculatus with Sin Nombre virus.

Data from naturally infected deer mice (Peromyscus maniculatus) were used to investigate vertical transmission of Sin Nombre virus (SNV) and SNV-specific antibody. The antibody prevalence in juvenile mice (14 g or less) was inversely proportional to the mass of the animal, with juvenile deer mice weighing less than 11 g most likely to be antibody positive (26.9%) and juvenile mice weighing between 13 and 14 g least likely to be antibody positive (12.9%). Although a significant sex bias in seropositivity was detected in adult deer mice, no significant sex bias in seropositivity was detected in juvenile animals. Ten juvenile deer mice were identified that had initially tested positive for SNV-specific immunoglobulin G (IgG) by enzyme-linked immunosorbent assay (ELISA) but had subsequently tested negative when recaptured as adults. SNV RNA was detected by reverse transcriptase PCR (RT-PCR) in the blood of ELISA-positive adult deer mice but not in the blood of ELISA-positive juveniles. One of the juvenile mice initially tested negative for SNV RNA but later tested positive when recaptured as an ELISA-positive adult. The RT-PCR results for that individual correlated with the disappearance and then reappearance of SNV-specific IgG, indicating that the presence of SNV RNA at later time points was due to infection with SNV via horizontal transmission. SNV-specific antibody present in both ELISA-positive juvenile and adult mice was capable of neutralizing SNV. Additionally, our data indicate that SNV is not transmitted vertically.

Age Factors↗

Antibodies to Rickettsia rickettsii in Peromyscus leucopus from a focus of Rocky Mountain spotted fever in Connecticut.

During 1980-1982, white-footed mice (Peromyscus leucopus) were captured in Newtown, Connecticut, an area where Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, is thought to be enzootic. An indirect microimmunofluorescence test identified specific antibodies to this organism in 16 of 237 (7%) sera: titration end points for 14 samples were relatively high (1:128-1:2048). Antibodies were detected in mice during 1980 and 1981 with monthly prevalences varying from 8 to 22%. These results suggest that P. leucopus may be involved in the ecology of R. rickettsii and that these rodents can be included along with other mammals to monitor spotted fever rickettsial infections in nature.

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Immunological memory is compromised by food restriction in deer mice Peromyscus maniculatus.

The immune system protects organisms against infection, but this protection presumably comes at a cost. Here, we asked whether food restriction would compromise the ability of an organism to generate an immune response on reexposure to an antigen, which would represent a functional cost of immunological memory. Immunological memory is generated when B and T lymphocytes sensitive to components of pathogens (i.e., antigens) proliferate after exposure and persist in circulation to hinder reinfection. To test the possibility that B cell memory, the component of the immune system responsible for antibody production, is expensive to maintain, secondary antibody production against a novel protein [keyhole limpet hemocyanin (KLH)] was compared in food-restricted and ad libitum-fed male deer mice (Peromyscus maniculatus). To determine whether compromised secondary antibody production was solely due to elevated corticosterone independent of resource availability, some food-restricted and ad libitum-fed mice were subjected to unpredictable, chronic (2 h/day) restraint. Mice fed 70% of their ad libitum diet 2 wk after primary antigen challenge produced approximately 95% less IgG against KLH after a second antigen challenge than mice fed ad libitum, even though all mice were fed ad libitum during the secondary antibody response period. Restraint had no effect on secondary IgG production in response to KLH, and corticosterone concentrations 1 day after food restriction did not differ between food-restricted and ad libitum-fed mice. Together, these data imply that secondary antibody responses and the benefits of immunological memory are energetically costly in this species.

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Social environment modulates photoperiodic immune and reproductive responses in adult male white-footed mice (Peromyscus leucopus).

Social cues may interact with photoperiod to regulate seasonal adaptations in photoperiod-responsive rodents. Specifically, photoperiod-induced adjustments (e.g., reproduction and immune function) may differ among individuals in heterosexual pairs, same-sex pairs, or isolation. Heterosexual cues may be more influential, based on their potential fitness value, than same-sex cues or no social cues. The present study examined the effects of pair (with a male or female) or individual housing on reproductive and immune responses in male white-footed mice (Peromyscus leucopus) maintained in long or short photoperiods. Female pairing did not affect reproductive responses in short-day males. In long days, however, the presence of a female increased both testosterone concentrations and testes mass compared with individually housed and male-paired mice, respectively. Short-day, individually housed males enhanced delayed-type hypersensitivity (DTH) responses compared with single-housed mice in long days, but all paired groups decreased DTH responses regardless of photoperiod. The lack of enhanced DTH response in male mice paired with females coincided with reduced circulating corticosterone concentrations in both photoperiod treatments. Together, these results suggest that social environment may have important modulatory effects on photoperiod-regulated immune responses in male white-footed mice.

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Short photoperiods reduce vascular endothelial growth factor in the testes of Peromyscus leucopus.

Testicular regression in rodents occurs after short-day exposure. Vascular support is withdrawn during regression, and, presumably, new angiogenesis is inhibited. Blood vessel growth and maintenance are regulated by paracrine factors, including vascular endothelial growth factor (VEGF). Reduced angiogenesis may contribute to the onset of photoperiod-induced regression; i.e., reduction of VEGF protein would be detected early during gonadal atrophy. Alternatively, loss of blood vessel maintenance may reflect reduced testicular volume. If true, VEGF would not be expected to decline until significant regression occurred. To discriminate between these hypotheses, white-footed mice (Peromyscus leucopus) were maintained in either long (LD 16:8) or short (LD 8:16) photoperiod. Immunohistochemical and Western analyses revealed high VEGF expression in Leydig and Sertoli cells in long-day housed males and reduced VEGF expression in short-day housed males. Reductions in VEGF preceded decreases in both seminiferous tubule diameter and spermatogenic activity by 6 wk and reduced testis mass by 8 wk, suggesting that changes in VEGF are not a consequence of gonadal regression and that VEGF may play a critical role in photoperiodic regulation of testicular function.

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