Serological evidence of hepatitis E virus infection in pigs and jaundice among pig handlers in Bangladesh.

Hepatitis E virus (HEV) is the most common cause of viral hepatitis in humans. Pigs may act as a reservoir of HEV, and pig handlers were frequently identified with a higher prevalence of antibodies to HEV. The objectives of this study were to identify evidence of HEV infection in pigs and compare the history of jaundice between pig handlers and people not exposed to pigs and pork. Blood and faecal samples were collected from 100 pigs derived from three slaughterhouses in the Gazipur district of Bangladesh from January to June, 2011. We also interviewed 200 pig handlers and 250 non-exposed people who did not eat pork or handled pigs in the past 2 years. We tested the pig sera for HEV-specific antibodies using a competitive ELISA and pig faecal samples for HEV RNA using real-time RT-PCR. Of 100 pig sera, 82% (n = 82) had detectable antibody against HEV. Of the 200 pig handlers, 28% (56/200) demonstrated jaundice within the past 2 years, whereas only 17% (43/250) of controls had a history of jaundice (p < .05). Compared to non-exposed people, those who slaughtered pigs (31% versus 15%, p < .001), reared pigs (37% versus 20%, p < .001), butchered pigs (35% versus 19%, p < .001) or involved in pork transportation (28% versus 13%, p < .001) were more likely to be affected with jaundice in the preceding 2 years. In multivariate logistic regression analysis, exposure to pigs (odds ratio [OR]: 2.2, 95% CI: 1.2-3.9) and age (OR: 0.97, 95% CI: 0.95-0.99) was significantly associated with jaundice in the past 2 years. Pigs in Bangladesh demonstrated evidence of HEV infection, and a history of jaundice was significantly more frequent in pig handlers. Identifying and genotyping HEV in pigs and pig handlers may provide further evidence of the pig's role in zoonotic HEV transmission in Bangladesh.

Efficiency of the Clinical Veterinary Diagnostic Practices and Drug Choices for Infectious Diseases in Livestock in Bangladesh.

As in most low-income countries, adequate laboratory facilities are not available in Bangladesh to assist veterinarians in diagnosing animal diseases. We aimed to determine the efficiency of veterinary diagnoses for two common ruminant diseases in Bangladesh: Peste des petits ruminants (PPR) and foot-and-mouth disease (FMD). We conducted the study from May 2009 to August 2010 in three government veterinary hospitals where veterinarians collected samples from sick livestock and recorded the presumptive diagnosis on the basis of clinical presentations. Samples were tested for PPR and FMD using real-time RT-PCR. We estimated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the presumptive diagnoses when compared to laboratory tests. We tested 539 goats for PPR and 340 cattle and goats for FMD. Our results indicate that the veterinarians' presumptive diagnoses were different from laboratory findings for both PPR (P < 0.05) and FMD (P < 0.05). The overall sensitivity of the presumptive clinical diagnoses was 54% (95% CI: 47-61%) while specificity was 81% (95% CI: 78-84%) compared to real-time RT-PCR tests. The kappa value obtained in our validation process for PPR (kappa: 0.25) and FMD (kappa 0.36) indicated a poor performance of the presumptive diagnoses. Most of the animals (93%) were treated with antibiotics. Our findings indicate that veterinarians can detect animals not infected with FMD or PPR but miss the true cases. The clinical competency of these veterinarians needs to be improved and access to laboratory diagnostic facilities could help veterinarians to improve the diagnostics and outcomes. The rational use of antibiotics by veterinarians in animals must be ensured.

Immunization with adenoviral-vectored tick salivary gland proteins (SALPs) in a murine model of Lyme borreliosis.

Prior exposure of vertebrate hosts to tick salivary proteins can induce specific immunity to tick infestation, as well as affording protection against tick-transmitted Borrelia burgdorferi infection in the mammalian host. Vaccination using an adenovirus expression system to deliver 4 tick salivary proteins (Ad-Salps) derived from Ixodes scapularis, Salp15, Salp25A, Salp25D, and Isac, was explored. Results indicate that vaccination with tick salivary proteins in an adenoviral vector can be used to modulate a Th1 response in the host and partially control spirochete load in immunized mice after infected tick challenge.

Association of human immune response to Aedes aegypti salivary proteins with dengue disease severity.

Dengue viruses (DENV; family Flaviviridae, genus Flavivirus) are transmitted by Aedes aegypti mosquitoes and can cause dengue fever (DF), a relatively benign disease, or more severe dengue haemorrhagic fever (DHF). Arthropod saliva contains proteins delivered into the bite wound that can modulate the host haemostatic and immune responses to facilitate the intake of a blood meal. The potential effects on DENV infection of previous exposure to Ae. aegypti salivary proteins have not been investigated. We collected Ae. aegypti saliva, concentrated the proteins and fractionated them by nondenaturing polyacrylamide gel electrophoresis (PAGE). By the use of immunoblots, we analysed reactivity with the mosquito salivary proteins (MSP) of sera from 96 Thai children diagnosed with secondary DENV infections leading either to DF or DHF, or with no DENV infection, and found that different proportions of each patient group had serum antibodies reactive to specific Ae. aegypti salivary proteins. Our results suggest that prior exposure to MSP might play a role in the outcome of DENV infection in humans.

Kinetics of Borrelia burgdorferi infection in larvae of refractory and competent tick vectors.

The acquisition of Borrelia burgdorferi by the larvae of competent and refractory ixodid ticks was assessed by quantitative polymerase chain reaction (PCR). Larvae were fed on infected mice, and the spirochete loads were determined during feeding and up to 93 d postfeeding. Amblyomma americanum (L.) was refractory to B. burgdorferi infection, with almost no detection of spirochete DNA during or postfeeding. In contrast, Ixodes scapularis Say supported high loads of spirochetes (10(3)-10(4) per larva). In Dermacentor variabilis (Say), B. burgdorferi uptake was reduced, with an average of 16 spirochetes per larvae acquired after 4 d of feeding, representing 1/195 of the counts in I. scapularis. However, during the first day postfeeding, the spirochete growth rate in D. variabilis reached 0.076 generations per hour, 7.7 times greater than the highest growth rate detected in I. scapularis. D. variabilis supported intense spirochete growth up to the fourth day postinfection, when the counts increased to an average of 282 spirochetes per larvae or 1/8.5 of the I. scapularis counts 4 d postfeeding. The kinetics of spirochete growth was unstable in D. variabilis compared with I. scapularis, and transmission of B. burgdorferi by D. variabilis could not be demonstrated. A cofeeding experiment indicated that I. scapularis feeding increased A. americanum spirochete uptake. These collective results indicate suboptimal conditions for B. burgdorferi uptake and colonization within A. americanum or the presence of anti-Borrelia factor(s) in this nonpermissive tick species.

Capillary feeding of specific dsRNA induces silencing of the isac gene in nymphal Ixodes scapularis ticks.

Ixodes scapularis transmits several pathogens including Borrelia burgdorferi. Bioactive compounds in tick saliva support tick feeding and influence pathogen transmission to the mammalian host. These studies utilized oral delivery of dsRNA to silence an anticomplement gene (isac) in I. scapularis nymphs. Silencing of isac significantly reduced fed-tick weight compared to delivery of control lacZ dsRNA, and immunoblots specific for FlaB protein indicated a reduction in spirochete load in isac-silenced infected nymphs. SDS-PAGE demonstrated that isac gene silencing affected expression of a number of salivary and non-salivary gland proteins in ticks. Finally, multiple isac cDNA homologues were cloned, and these may represent a new gene family coexpressed during tick feeding. This work presents a novel oral delivery approach for specific gene silencing in I. scapularis nymphs and characterizes the effect of isac on blood-feeding in an attempt to block transmission of B. burgdorferi.

Differential infectivity of the Lyme disease spirochete Borrelia burgdorferi derived from Ixodes scapularis salivary glands and midgut.

Blood fed nymphal Ixodes scapularis Say infected with Borrelia burgdorferi were dissected to obtain salivary gland and midgut extracts. Extracts were inoculated into C3H/HeJ mice, and ear, heart, and bladder were cultured to determine comparative infectivity. Aliquots of extracts were then analyzed by quantitative polymerase chain reaction to determine the number of spirochetes inoculated into mice. A comparative median infectious dose (ID50) was determined for both salivary gland and midgut extract inoculations. Our data demonstrated a statistically significant difference (P < 0.002) in the ID50 derived from salivary gland (average = 18) versus midgut (average = 251) extracts needed to infect susceptible mice. A rationale for the differential infectivity of salivary and midgut derived spirochetes is discussed.

Borreliacidal activity of saliva of the tick Amblyomma americanum.

Amblyomma americanum (Linneaus) (Acari: Ixodidae), an important tick vector of human and animal disease, is not a competent vector of the bacterial agent of Lyme disease, Borrelia burgdorferi, although its range overlaps the geographical distribution of Lyme disease within the United States. A possible mechanism that could prevent acquisition of B. burgdorferi spirochetes from infected hosts is the toxic effect of A. americanum saliva on B. burgdorferi. The data presented here indicate that after 24 and 48 h of exposure to A. americanum saliva, significantly fewer B. burgdorferi were alive compared to treatment controls as assessed by spirochete motility under dark-field microscopy and resistance to the dead stain, propidium iodide. After 48 h, fewer than 13% of saliva-exposed B. burgdorferi were alive. In contrast, significantly more B. burgdorferi exposed to Ixodes scapularis (Acari: Ixodidae) saliva survived after 24 or 48 h compared to A. americanum saliva or treatment controls.

Three multiplex assays for detection of Borrelia burgdorferi sensu lato and Borrelia miyamotoi sensu lato in field-collected Ixodes nymphs in North America.

Two hundred fifty New Jersey field-collected Ixodes scapularis Say ticks and 17 Colorado Ixodes spinipalpis Hadwen & Nuttall ticks were tested using three separate multiplex real-time polymerase chain reaction (PCR) assays. One assay targets the rrs-rrlA IGS region of Borrelia spp. to detect Borrelia burgdorferi sensu lato (s.l.) and Borrelia miyamotoi s.l. The second assay targets the ospA region of B. burgdorferi s.l. to detect B. burgdorferi sensu stricto (s.s.), Borrelia bissettii, and Borrelia andersonii. The final assay targets the glpQ region of B. miyamotoi s.l. to differentiate B. miyamotoi LB-2001 and Borrelia lonestari. A testing scheme combining these tests yielded 18% of tested I. scapularis ticks surveyed from New Jersey positive for B. burgdorferi s.s., 3.2% I. scapularis ticks positive for B. miyamotoi LB-2001, and 41.2% I. spinipalpis ticks positive for B. bissettii surveyed from Colorado.

Sustained-release formulation of doxycycline hyclate for prophylaxis of tick bite infection in a murine model of Lyme borreliosis.

The prophylactic potential of a single injection of sustained-release doxycycline hyclate (Atridox) was compared to that of a single oral dose of doxycycline hyclate in a murine model of Lyme borreliosis. Prophylaxis, as measured by the lack of cultivable spirochetes and demonstrable pathology, was noted for 43% of orally treated mice; in contrast, the sustained-release doxycycline hyclate completely protected mice from infection and resultant pathology.

How much pilocarpine contaminates pilocarpine-induced tick saliva?

Pilocarpine is often applied or injected into ticks to induce salivation, and the resulting saliva used to test for various pharmacological, biochemical and immunological activities. To measure the amount of pilocarpine in pilocarpine-induced tick saliva, an HPLC-MS/MS method, based on capillary strong cation exchange chromatography online with an ion trap mass spectrometer, was used to measure pilocarpine in the pg to ng range. Results indicate large concentrations of pilocarpine in Ixodes scapularis Say and Amblyomma americanum (Linnaeus) (Acari: Ixodidae) saliva, ranging from 3 to 50 mm. Due to the known effects of pilocarpine on smooth muscle and immune cells, appropriate controls are proposed and discussed for proper interpretation of results using this saliva preparation.

Coinoculation of Borrelia spp. with tick salivary gland lysate enhances spirochete load in mice and is tick species-specific.

C3H/HeN mice were inoculated with 10(6) spirochetes, either Borrelia burgdorferi strain N40 or the Portuguese strain of B. lusitaniae, PotiB2. Mice receiving spirochetes coinoculated with salivary gland lysate (SGL) demonstrated significantly higher spirochete loads in target organs as measured by quantitative real-time polymerase chain reaction. This effect was tick dependent, in that Ixodes ricinus SGL specifically enhanced B. lusitaniae load, whereas I. scapularis SGL specifically increased B. burgdorferi N40 load, but did not significantly affect the dissemination of B. lusitaniae. Protein profile analysis indicated at least 5 major protein differences between I. scapularis and I. ricinus SGL, which can possibly account for this specific tick-spirochete interaction.

Use of a sentinel host system to study the questing behavior of Ixodes spinipalpis and its role in the transmission of Borrelia bissettii, human granulocytic ehrlichiosis, and Babesia microti.

Ixodes spinipalpis maintains Borrelia bissettii spirochetes in Colorado in a cycle involving wood rats and deer mice. This tick has been described as nidicolous, remaining either attached to its rodent hosts or in the rodent nest. Nidicolous ticks pose little risk of pathogen transmission to humans if they do not actively quest for hosts. To investigate the questing potential of I. spinipalpis, sentinel mice were placed in an area where I. spinipalpis had been commonly found on wood rats and deer mice. Concurrently, wild rodent populations were trapped and analyzed for Lyme disease spirochetes, the agent of human granulocytic ehrlichiosis (aoHGE), and Babesia microti. A total of 122 I. spinipalpis larvae and 10 nymphs were found on 19% of 244 sentinel mice. In addition, 4 sentinel mice became infested with Malaraeus telchinus or Orchopeas neotomae fleas. Questing I. spinipalpis were positively associated with woody shrubs and negatively associated with sunny and grassy areas. Four sentinel mice became infected with aoHGE after having been fed upon only by I. spinipalpis larvae. One sentinel mouse became infected with B. bissettii after having an I. spinipalpis nymph feed on it, and one sentinel mouse became coinfected with aoHGE and B. bissettii after it was fed upon by a single I. spinipalpis nymph. These sentinel mouse conversions suggest the possibility that the aoHGE is transovarially transmitted by I. spinipalpis, and that I. spinipalpis is capable of simultaneously transmitting B. bissettii and the aoHGE. The findings that I. spinipalpis quest away from rodent nests and will attach to and infect sentinel mice may be of public health importance. It suggests the potential transmission of the agents of human granulocytic ehrlichiosis and Lyme disease to other hosts by I. spinipalpis, in regions of the western United States where Ixodes pacificus is not found.

Use of quantitative PCR to measure density of Borrelia burgdorferi in the midgut and salivary glands of feeding tick vectors.

Quantitative real-time PCR was used to assay spirochetes in feeding ticks. Spirochetes in tick midguts increased sixfold, from 998 per tick before attachment to 5,884 at 48 h of attachment. Spirochetes in tick salivary glands increased >17-fold, from 1.2 per salivary gland pair before feeding to 20.8 at 72 h postattachment. The period of the most rapid increase in the number of spirochetes in the salivary glands occurred from 48 to 60 h postattachment; this time period coincides with the maximal increase in transmission risk during nymphal tick feeding.

Borrelia lonestari DNA in adult Amblyomma americanum ticks, Alabama.

Polymerase chain reaction analysis of 204 Amblyomma americanum and 28 A. maculatum ticks collected in August 1999 near the homes of patients with southern tick-associated rash illness and in control areas in Choctaw County, Alabama, showed Borrelia lonestari flagellin gene sequence from two adult A. americanum. The presence of B. lonestari in A. americanum ticks from Alabama suggests that this suspected pathogen may be widespread in the southeastern United States.

An analysis of spirochete load, strain, and pathology in a model of tick-transmitted Lyme borreliosis.

Four laboratory-grown, low-passage isolates of Borrelia burgdorferi sensu stricto, B31, JD-1, 910255, and N40, were incorporated into Ixodes scapularis ticks to examine the pathogenesis of these isolates in mice after tick transmission. All isolates induced multifocal, lymphoid nodular cystitis, subacute, multifocal, necrotizing myocarditis, and a localized periostitis and arthritis of the femorotibial joint 6-18 weeks after tick infestation. In terms of the number of mice that demonstrated pathology in bladder, heart, and joint, the highest incidence of lesions occurred 12 weeks after tick bite. Utilizing the Taqman quantitative polymerase chain reaction (q-PCR) fluorogenic detection technology to amplify a conserved region of the flagellin gene, a trend was demonstrated between the number of spirochetes in tissue with duration of pathology. The q-PCR assay developed for this study was sensitive and could reliably measure as few as 1 to 10 spirochetes in the target tissues tested. A higher percentage of B31- and N40-infected mice (92 and 100%, respectively) developed myocarditis than JD-1- or 910255-infected mice (67 and 46%, respectively) 12 weeks after tick bite. The amount of spirochetal DNA that could be amplified for heart at this time point was not statistically different between isolates, indicating a difference in virulence between B31 and N40 relative to JD-1 and 910225. N40-infected mice demonstrated a significantly higher spirochete load (an average of 1.23 spirochetes/mg of tissue, p = 0.045) in femorotibial joints 18 weeks after infection, with 60% of these mice maintaining lesions compared with those infected with B31 (13%), JD-1 (25%), or 910255 (50%), which averaged <0.5 spirochetes/mg of tissue. This mouse model of Lyme borreliosis, including the ability to monitor lesion development and spirochete load, can facilitate the testing of therapeutic regimens for the later stages of tick-transmitted Lyme disease and help investigate aspects of the immunopathogenesis of lesion development.

Coinfection with Borrelia burgdorferi and the agent of human granulocytic ehrlichiosis suppresses IL-2 and IFN gamma production and promotes an IL-4 response in C3H/HeJ mice.

Previously we demonstrated that Borrelia burgdorferi transmission by Ixodes scapularis suppressed IL-2 and IFN gamma production and promoted IL-4 production in mice. The present studies were conducted to determine whether coinfection with the human granulocytic ehrlichiosis (HE) agent would promote a Th2 cytokine response in mice. Transmission to the spleen of the agent of human granulocytic ehrlichiosis (aoHGE) and B. burgdorferi occurred 4 and 7 days, respectively, after tick infestation. Coinfection synergized to suppress splenic IL-2 production 7-14 days after tick infestion. Transmission of B. burgdorferi or aoHGE alone significantly decreased splenic IFN gamma 4-7 days after tick infestation, while coinfection suppressed IFN gamma production 7-14 days after tick infestation. Splenic IL-4 production was significantly increased 4 days after coinfection, and by day 10, aoHGE plus B. burgdorferi induced greater splenic IL-4 (57.2 pg/ml, 348% of control values) than either organism transmitted alone (aoHGE, 22.7 pg/ml, B. burgdorferi, 25.1 pg/ml). Coinfection enhanced expansion of splenic T cells, CD4+ lymphocytes and B cells while decreasing CD8+ T cells. These data demonstrate that aoHGE and B. burgdorferi, when cotransmitted, suppress a systemic IL-2 and IFN gamma response, while strongly promoting systemic IL-4 production in the susceptible host. The antigen(s) responsible for this polarization are unknown and will be the subject of future studies.

Borrelia isolates in Northern Colorado identified as Borrelia bissettii.

Previous work described Borrelia burgdorferi sensu lato group DN127 as a new genospecies, Borrelia bissettii, and prompted the present study to identify the Borrelia spp. that exist in northern Colorado. To determine the genospecies present, we analyzed two specific intergenic spacer regions located between the 5S and 23S and the 16S and 23S ribosomal genes. Phylogenetic analysis of the derived sequences clearly demonstrated that these isolates, originating from rodents captured in the foothills of northern Colorado, diverged from B. burgdorferi sensu stricto by 5 to 5.5% and were members of the new genospecies B. bissettii.

Transmission of the agent of human granulocytic ehrlichiosis by Ixodes spinipalpis ticks: evidence of an enzootic cycle of dual infection with Borrelia burgdorferi in Northern Colorado.

Previous work described an enzootic cycle of Borrelia burgdorferi sensu lato (hereafter referred to as B. burgdorferi) maintained by the rodent Neotoma mexicana and the tick Ixodes spinipalpis in northern Colorado. We investigated the incidence of coinfection among rodents with the agent of human granulocytic ehrlichiosis (aoHGE). aoHGE was detected in 23.5% of 119 rodent spleens examined. Biopsy results indicated that 78 (65.5%) of the 119 rodents were positive for B. burgdorferi, whereas 22 (78.5%) of the 28 animals that harbored aoHGE were also infected with B. burgdorferi. In 14 of 25 I. spinipalpis tick pools, aoHGE was detected by amplifying both the 16s rRNA and p44 gene of aoHGE. The ability of I. spinipalpis to transmit aoHGE was examined in C3H/HeJ mice. aoHGE was detected in their blood 5 days after I. spinipalpis infestation. This study confirms that both B. burgdorferi and aoHGE can be transmitted by I. spinipalpis ticks and that there is a high incidence of coinfection in rodents, predominantly Peromyscus maniculatus and N. mexicana, that inhabit the foothills of northern Colorado.

Babesia microti and Borrelia bissettii transmission by Ixodes spinipalpis ticks among prairie voles, Microtus ochrogaster, in Colorado.

An endemic transmission cycle of Babesia microti was discovered in Colorado in the foothills of the Rocky Mountains. B. microti were found by PCR in 4 of 25 Ixodes spinipalpis tick pools tested (a 3.2 % minimum infection rate) and in 87% (13 of 15) of Microtus ochrogaster (the prairie vole) spleen and blood samples. Using naturally infected I. spinipalpis collected from wild-caught M. ochrogaster as vectors, B. microti and Borrelia bissettii were successfully transmitted to laboratory-born M. ochrogaster. Neither I. spinipalpis, nor M. ochrogaster (the prairie vole) have been previously reported as a vector or a reservoir host of B. microti. Unlike the east coast of the United States where Peromyscus leucopus is an important reservoir for B. microti, evidence for Peromyscus spp. (neither P. maniculatus nor P. difficilis) as B. microti reservoirs was not found in this study.