Vector-Borne and Zoonotic Diseases

Borrelia burgdorferi, the bacterium that causes Lyme disease, is transmitted to a susceptible host by Ixodes spp. tick bites. However, there is uncertainty whether B. burgdorferi are shed from ticks by the fecal route. In this study, B. burgdorferi–infected ticks were fed on mice while confined to a certain area of the skin by a capsule. During and after feeding, tick feces were collected and placed in Barbour-Stoenner-Kelley (BSK)-II media for cultivation and in sterile water for polymerase chain reaction (PCR) analysis. Although none of the tested samples were culture positive for B. burgdorferi, all but one of the fecal DNA samples from infected ticks were PCR positive. These results indicated that B. burgdorferi were shed from feeding ticks during defecation and suggest that the spirochetes did not remain viable once exposed to the outside environment. This finding has important ramifications for investigators interpreting B. burgdorferi–specific PCR results when conducting tick transmission experiments.

Tick-borne relapsing fever (TBRF) is endemic in Africa and Eurasia and attributed to different Borrelia species. In the Central Asia and Middle Eastern countries, TBRF is caused mainly by Borrelia persica; however, other Borrelia species such B. microtti, B. latyschewii, B. baltazardi, and B. caucasica have also been described. The classic taxonomy of Borrelia spp. is based on the cospeciation concept that is very complex and rather confusing. In this study, we report two DNA-based methods to discriminate B. persica and B. microtti, the two main prevalent species in the region. Molecular typing of the species was performed using (i) restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified fragments of either 16S-rRNA or glpQ genes, and (ii) species-specific PCR of glpQ gene. Sequence analyses of the data obtained in this study indicate that the glpQ gene is more variable than 16S-rRNA (6.9% vs. 1.2%); thus glpQ is a more useful marker for discrimination of B. persica from B. microtti. The 16S-rRNA fragment comprises only one useful species-specific restriction site (TaqI), whereas the glpQ fragment includes several species-specific restriction sites and its digestion by DraI, TaqI, EcoRV, HinfI, or SspI results in distinctively different PCR–restriction fragment length polymorphism patterns for the two species. Further, the species-specific primers amplified a 253-bp fragment for B. persica and a 451-bp one for B. microtti. Phylogenetic analysis of the data revealed that B. microtti and B. persica are associated to the African and new world RF agents, respectively. This study demonstrates that both typing methods are simple, sensitive, and fast, and that they allow one to differentiate between B. persica and B. microtti. This could prove that both methods are important and useful in monitoring of TBRF disease in endemic areas.

To identify potential vector species of scrub typhus in the Republic of Korea (ROK), chigger mites were harvested from wild rodents captured at nine localities in October 2005. The bodies of the chigger mites were individually punctured with a fine pin, squeezed out internal contents, and examined for Orientia tsutsugamushi DNA by nested polymerase chain reaction. The exoskeleton of associated chiggers was mounted on glass slides with polyvinylalcohol (PVA) medium for identification. Among 830 individuals belonging to 4 genera and 14 species, O. tsutsugamushi was detected from 22 chiggers of six species, with an overall infection rate of 2.7%. The infection rate was highest for Leptotrombidium palpale (5.3%), followed by Neotrombicula japonica (4.3%), Leptotrombidium scutellare (3.7%), Leptotrombidium orientale (3.6%), Eushoengastia koreaensis (1.9%), and Leptotrombidium pallidum (1.5%). This study first reported O. tsutsugamushi infection from N. japonica and E. koreaensis larvae in the ROK. The population densities of L. pallidum (33.4 chiggers/rodent), historically confirmed as a primary vector of scrub typhus in the ROK, were high, whereas its infection rate was relatively low (1.5%). However, E. koreaensis was only collected from 154 individuals at seven collection sites and its infection rate was demonstrated relatively high (mean 1.9%). Additional studies are needed to determine the role of vector species in the epidemiology of scrub typhus.

Streptococcus suis serotype 2 (S. suis S2) is able to cause human infections ranging from superficial wounded skin infections to severe invasive infections such as meningitis and streptococcal toxic shock-like syndrome. During its infection cycle, S. suis S2 must acclimatize itself to temperature shift. Herein, a whole-genome DNA microarray was used to investigate the global transcriptional regulation of an invasive strain of S. suis S2 grown to late-exponential phase at 29°C or 40°C relative to 37°C. The differentially regulated genes that were detected included those encoding virulence factors, antigenic proteins, ATP-binding-cassette transporters, and proteins of unknown functions. Our data provided a global profile of gene transcription induced by temperature alteration and shed light on some unforeseen lines for further pathogenesis investigation.

A pilot study was conducted to determine whether existing human or canine strains of Anaplasma phagocytophilum would reproduce clinical disease in experimentally inoculated dogs similar to dogs with naturally acquired granulocytic anaplasmosis. Six hounds were inoculated intravenously with one human and two canine strains of A. phagocytophilum that were propagated in vitro in HL-60 cells or in infected autologous neutrophils. Infected dogs were monitored for lethargy, anorexia, petechiae, lymphadenopathy, and fever. Dogs were assessed for complete blood count (CBC), serum chemistry, and serology (IFA and SNAP^® 4Dx^®); for A. phagocytophilum blood load by quantitative polymerase chain reaction; and for cytokine production. Prominent clinical signs were generalized lymphadenopathy and scleral injection; only one dog developed fever lasting 4 days. Notable laboratory alterations included sustained leukopenia and thrombocytopenia in all dogs. A. phagocytophilum morulae were noted in blood between days 10 and 11, although all dogs retained A. phagocytophilum DNA in blood through day 60. All dogs seroconverted by days 10–15 by IFA, and by days 17–30 by SNAP 4Dx; cytokine analyses revealed 10-fold increases in interleukin-2 and interleukin-18 in the neutrophil-propagated 98E4 strain-infected dog. All A. phagocytophilum strains produced infection, although canine 98E4 strain reproduced clinical signs, hematologic changes, and inflammatory cytokine elevations most consistent with granulocytic anaplasmosis when recognized clinically. Therefore, this strain should be considered for use in future studies of A. phagocytophilum canine infection models.

Domestic and wild rodents known as the most abundant and diversified order of mammals have a key role in the ecological food chain and also in the transmission of parasites and pathogens to other animals. While foraging on the ground, they can get infected by Toxoplasma gondii, a protozoan parasite, which is the causative agent of toxoplasmosis. Therefore, they serve as intermediate hosts of T. gondii and can transmit it to their predators. To assess their role in the maintenance of T. gondii lifecycle in Thailand, we sampled rodents in a range of biotopes representative of the high biodiversity and conducted a serological survey with latex agglutination test to detect anti-T. gondii antibodies. Overall, 21 of 461 (4.6%) rodents had diagnostically significant antibody titers (cutoff, 1:64). Every species with at least 37 individuals captured tested positive, confirming the wide range of potential mammalian hosts of toxoplasmosis. None of the ecological traits (sex, maturity, morphology, season, or habitat) was found significant to predict the susceptibility to T. gondii both univariately and in a multivariate analysis. However, high prevalences were reported in either forested or anthropized areas. This survey constitutes the first confirmed serological investigation of T. gondii in rodents in Thailand. The rarity of both domestic and wild felids in Thailand emphasizes the importance of rodents in maintaining T. gondii, and questions the involvement of other carnivores in the life cycle.

The aim of this study was to evaluate the prevalence of Leishmania infantum infection within a feline population by serologic and molecular methods and to identify associated risk factors. One hundred five cats living outdoors were studied. Sera were tested for IgG antibodies against L. infantum, Toxoplasma gondii, and feline immunodeficiency virus (FIV) and for the detection of feline leukemia virus (FeLV) p27 antigen by enzyme-linked immunosorbent assay (ELISA). L. infantum real-time polymerase chain reaction (PCR) was performed on DNA extracted from blood. L. infantum and T. gondii seroprevalence rates were 13.2% and 55.2%, respectively. The prevalence of L. infantum by PCR was 8.7%. The total rate of L. infantum infection derived from seroreactivity and/or positive PCR was 15.4%. Serology and PCR results were positively associated, and moderate agreement (kappa = 0.489) was found between Leishmania ELISA and PCR. No statistical association was found between positive Leishmania PCR results and gender, clinical status, or T. gondii seropositivity. Six of the 105 cats (5.7%) displayed clinical signs compatible with feline cutaneous leishmaniosis, and 4 out of these 6 cats (66.7%) were found to have Leishmania infection by means of serology and/or PCR. Leishmania seropositivity was associated with clinical signs of feline cutaneous leishmaniosis (p = 0.029). The prevalence of FeLV p27 antigen was 16.2% (17/105) and of FIV antibody was 20.9% (22/105), with coinfection found in 9.5% (10/105) of the cats. Leishmania ELISA seroreactivity and positive PCR results were statistically associated with FeLV infection and with coinfection of both retroviruses but not with a positive FIV status. The high seroprevalence and molecular rates of Leishmania infection observed indicate that cats are frequently infected with L. infantum, and the association with FeLV suggests a potential role for this retrovirus in feline Leishmania infection in endemic areas.

Leishmaniasis is endemic in the Middle East, and both cutaneous and visceral forms are reported from the region ranging from the Levant to Afghanistan. The potential and proven phlebotomine sand fly vectors and reservoir hosts of the Leishmaniases species in Afghanistan, Iran, Iraq, Israel, Jordan, Lebanon, Saudi Arabia, Syria, Turkey, and Yemen are described. This region has seen a movement of populations across the area, due to both military and civilian strife. Refugees, armed forces, and multi-national contractors are particularly at risk to acquire this disease. There has been an upsurge in Leishmaniasis research, especially as new foci are exposed and the need to protect the naïve populations moving into endemic areas becomes a public health priority. New sand fly vectors and animal reservoirs have been discovered while novel control methods are being evaluated. Modern molecular techniques are now being used more routinely and revealing some unusual findings. The aim of this review is to collate the most recent data on the burden of the disease, diagnostic applications, eco-epidemiology of vectors, and reservoir hosts, and how the control projects have been developing in the Middle East.

An ethnopharmacological investigation was undertaken on Western Ghats plants traditionally used to treat malaria; 50 plants were very carefully selected from total of 372 plants, and 216 extracts were prepared and tested for in vivo antiplasmodial activity alone and in combination with chloroquine (CQ) against CQ-tolerant Plasmodium berghei (strain NK65). In in vivo antiplasmodial activity when plant extract alone is used, 81 extracts (or 37.5%) gave 52.90% significant parasitemia inhibition on day 4 postinfection and 39 extracts (or 18%) gave 41–89% mouse survival on day 9 postinfection. In combination with CQ on day 11 postinfection, 103 extracts (or 47.68%) gave mouse survival rate of 92% and on day 14 gave maximum mouse survival up to 70–79%. The fact that these activities were up to fourfold higher with CQ mostly resulted in longer mouse survival because of significant parasitemia inhibition. Our investigation have confirmed that above 70% of the plant extracts showed moderate to high in vivo antimalarial activity when used alone and in combination with CQ, and most of the extracts showed border line to good synergistic activity.

The decrease in western equine encephalomyelitis virus (WEEV; Togaviridae, Alphavirus) activity in North America over the past 20–30 years has prompted research to determine if there have been concurrent declines in virulence. Six (WEEV) strains isolated from Culex tarsalis mosquitoes from California during each of the six preceding decades failed to show a consistent declining temporal trend in virus titer using mosquito (C6/36), avian (duck embryo fibroblast), or mammalian (Vero) cells, results similar to our recent in vivo studies using birds and mosquitoes. Titers measured by Vero cell plaque assay were consistently highest on mosquito cell culture, followed by avian and mammalian cell cultures. Similar to previous in vivo results in house sparrows and mice, titers for the IMP181 strain isolated in 2005 were significantly lower in both avian and mammalian cells. Real-time monitoring of changes in cell growth measured by electrical impedance showed consistent differences among cell types, but not WEEV strains. Collectively, these in vitro results failed to explain the decrease in WEEV enzootic and epidemic activity. Results with the IMP181 strain should be verified by additional sequencing, cell growth, and pathogenesis studies using concurrent or 2006 isolates of WEEV from California.

Yellow fever (YF) is a serious public health problem in Bolivia since at least the 19th century. Surprisingly, very limited information has been made available to date regarding the genetic characterisation and epidemiology of Bolivian YF virus (YFV) strains. Here, we conducted the genetic characterization of 12 human isolates of YFV collected in Bolivia between 1999 and 2008, by sequencing and analysis of two regions of the viral genome: a fragment encoding structural proteins “PrM” (premembrane and envelope) and a distal region “EMF,” spanning the end of the virus genome. Our study reveals a high genetic diversity of YFV strains circulating in Bolivia during the last decade: we identified not only “Peruvian-like” genotype II viruses (related to previously characterized Bolivian strains), but also, for the fist time, “Brazilian-like” genotype I viruses. During the complete period of the study, only cases of “jungle” YF were detected (i.e., circulation of YFV via a sylvatic cycle) with no cluster of urban cases. However, the very significant spread of the Aedes aegypti mosquito across Bolivian cities threatens the country with the reappearance of an urban YFV transmission cycle and thus is required a sustained epidemiological surveillance.

West Nile virus (WNV) is a neurotropic mosquito-transmitted flavivirus that in Eurasia, Africa, and the Americas primarily affects birds and secondarily other vertebrates. WNV has caused frequent massive episodes of wild bird mortality during its expansion throughout the Americas, and has become a regulating factor in the population dynamics of many wild bird species. On the other hand, WNV-related mortalities in wild birds have rarely been reported in the Mediterranean Basin despite its well-documented circulation, and only sporadic outbreaks in horses have been documented. The causes underlying this contrasting epidemiological pattern have never been properly described. An initial suggestion is that Mediterranean and American strains possess different pathogenicities, whereas an alternative view proposes that WNV-related disease and mortalities may have been overlooked in Europe. To test these hypotheses, between 2004 and 2006 in southern Spain we sampled tissue from 119 wild bird carcasses to detect WNV and other flaviviruses, as well as blood from 227 wild birds arriving in wildlife rehabilitation centers to test for WNV seroprevalence. No flavivirus was found in the tissue samples. The prevalence of WNV-neutralizing antibodies was 2.2%, similar to that of 800 healthy birds of the same species that were captured in the field. Our results suggest that WNV circulation during the study period did not result in any detectable effects in terms of bird morbidity or mortality.

Arboviruses are frequently associated with outbreaks in humans and represent a serious public health problem. Among the Brazilian arboviruses, Mayaro virus, Dengue virus (DENV), Yellow Fever virus, Rocio virus, Saint Louis Encephalitis virus (SLEV), and Oropouche virus are responsible for most of human cases. All these arboviruses usually produce undistinguishable acute febrile illness, especially in the acute phase of infection. In this study we investigated the presence of arboviruses in sera of 519 patients presenting acute febrile illness, during a dengue outbreak in São José do Rio Preto City (São Paulo, Brazil). A multiplex-nested RT-polymerase chain reaction assay was applied to detect and identify the main Brazilian arboviruses (Flavivirus, Alphavirus, and Orthobunyavirus genera). The molecular analysis showed that 365 samples were positive to DENV-3, 5 to DENV-2, and 8 to SLEV. Among the positive samples, one coinfection was detected between DENV-2 and DENV-3. The phylogenetic analysis of the SLEV envelope gene indicated that the virus circulating in city is related to lineage V strains. These results indicated that during that large DENV-3 outbreak in 2006, different arboviruses cocirculated causing human disease. Thus, it is necessary to have an efficient surveillance system to control the dissemination of these arboviruses in the population.

An ecological assessment of reservoir species was conducted in a rural area (Jaborá) in the mid-west of the state of Santa Catarina in southern Brazil, where hantavirus pulmonary syndrome is endemic, to evaluate the prevalence of hantavirus infection in wild rodents. Blood and tissue samples were collected from 507 rodents during seven field trips from March 2004 to April 2006. Some of the animals were karyotyped to confirm morphological identification. Phylogenetic reconstructions of rodent specimens, based on the mitochondrial DNA cytochrome b gene sequences, were also obtained. Hantavirus antibody was found in 22 (4.3%) of the 507 rodents: 5 Akodon montensis, 2 Akodon paranaensis, 14 Oligoryzomys nigripes, and 1 Sooretamys angouya. Viral RNAs detected in O. nigripes and A. montensis were amplified and sequenced. O. nigripes virus genome was 97.5% (nt) and 98.4% (nt) identical to sequences published for Araucaria (Juquitiba-like) virus based on N and G2 fragment sequences. Viral sequences from A. montensis strain showed 89% and 88% nucleotide identities in a 905-nt fragment of the nucleocapsid (N) protein-coding region of the S segment when it was compared with two other Akodontine rodent-associated viruses from Paraguay, A. montensis and Akodon cursor, respectively. Phylogenetic analysis showed the cocirculation of two genetic hantavirus lineages in the state of Santa Catarina, one from O. nigripes and the other from A. montensis, previously characterized in Brazil and Paraguay, respectively. The hantavirus associated with A. montensis, designed Jaborá virus, represents a distinct phylogenetic lineage among the Brazilian hantaviruses.

Modifications of natural habitat in peridomestic rural areas could affect original rodent community composition, diversity, and evenness. In zoonoses such as hantavirus pulmonary syndrome, the presence of a diverse community can dilute the impact of the principal reservoir, reducing risk to humans. The goal of this study was to examine rodent community composition, abundance of Andes virus (ANDV) host (Oligoryzomys longicaudatus), ANDV prevalence, and temporal variability associated with rural peridomestic settings in Patagonia, Argentina. We trapped rodents in peridomestic settings and nearby sylvan areas for 2 years. The numerically dominant species differed between peridomestic and sylvan settings. O. longicaudatus was the most abundant species in peridomestic settings (>50% of individuals). Diversity and evenness in peridomestic settings fluctuated temporally, with an abrupt decline in evenness coinciding with peaks in ANDV prevalence. The probability of finding an ANDV-positive mouse in peridomestic settings was 2.44 times greater than in sylvan habitats. Changes in rodent communities in peridomestic settings may increase the probability for human exposure to ANDV because those settings promote the presence of O. longicaudatus with high ANDV antibody prevalence. High O. longicaudatus relative abundance in an unstable community associated with peridomestic settings may favor intraspecific contact, leading to a higher probability of virus transmission.