Vector competence of Ochlerotatus trivittatus (Coquillett), Aedes albopictus (Skuse), and Culex pipiens (L) (Diptera: Culicidae) for West Nile virus

West Nile virus (WNV) was recognized in North America in 1999. Avian species are the primary reservoir of the virus and can develop viremias with titers higher than 1010.0 CID50s/ml. Culex species of mosquitoes are the primary amplifying vectors. Many species of mammals are also susceptible to WNV but develop viremias that seldom exceed 105.0 CID50s/ml. Mammals may play a significant role in WNV ecology if mosquitoes that feed on them during periods of viremia can be infected and transmit the virus. The primary objective of this study was to determine vector competency of Ochlerotatus trivittatus (Coq.) a potential bridge vector that feeds primarily on mammals including humans and occasionally on birds. Susceptibility of Oc. trivittatus to WNV was compared to that of Aedes albopictus (Skuse) a known bridge vector, and Culex pipiens (L.), a primary amplifying vector by determining infection rates after feeding mosquitoes on chicks with blood meal titers (BMTs) of 102.5 to 1010.0 CID50s/ml. The ability of Oc. trivittatus to transmit WNV was determined by comparing transmission rates (%) of Oc. trivittatus, Ae. albopictus and Cx. pipiens by the capillary tube method following infection by blood meals with titers ranging from 102.5 to 109.5 CID50s/ml. Susceptibility of Oc. trivittatus and Cx. pipiens to WNV was essentially the same but greater than Ae. albopictus. The lowest infective BMTs for Oc. trivittatus, Ae. albopictus and Cx. pipiens were 104.5 , 105.5, and 104.5 CID50s/ml. The 50% infective BMTs for the 3 species were 106.0 10 6.6, and 106.2 CID50s/ml. Transmission rates of Oc. trivittatus, Ae. albopictus, and Cx. pipiens after blood meals with titers higher than 107.0 CID 50s/ml were 41.7, 72.4 and 46.8%. The lowest BMTs that resulted in transmission by the 3 species were 105.5, 107.0, and 10 5.5 CID50s/ml. These observations suggest that Oc. trivittatus might play a more significant role than Ae. albopictus in maintaining WNV in populations of mammals which typically develop low levels of viremia

West Nile Virus Viremia in Eastern Chipmunks (Tamias striatus) Sufficient for Infecting Different Mosquitoes

Chipmunks might play a role in enzootic WNV cycles and be an amplifying host for mosquitoes that infect humans

Diversity of Anaplasma and novel Bartonella species in Lipoptena fortisetosa collected from captive Eld’s deer in Thailand

Lipoptena insects are important ectoparasites of cervids and may affect humans that are incidentally bitten. The presence of zoonotic pathogen DNA, such as Anaplasma, and Bartonella, raises the importance of Lipoptena insects in veterinary and human medicine. Eld’s deer (Rucervus eldii thamin), an endangered wild ruminant in Thailand, are bred and raised in the open zoo. The semi-wild zoo environment suggests ectoparasite infestation and potential risk for mechanical transmission of pathogens to visitors, zoo workers, or other animals. However, epidemiology knowledge of pathogens related to endangered wild ruminants in Thailand is limited. This study aims to determine the prevalence and diversity of Anaplasma and Bartonella in the L. fortisetosa collected from captive Eld’s deer in Chon Buri, Thailand. Of the 91 Lipoptena DNA samples obtained, 42 (46.15%) and 25 (27.47%) were positive for Anaplasma and Bartonella by molecular detection, respectively. Further, 42 sequences of Anaplasma (4 nucleotide sequence types) showed 100% identity to those detected in other ruminants and blood-sucking ectoparasites. Twenty-five sequences of Bartonella (8 nucleotide sequence types) showed 97.35–99.11% identity to the novel Bartonella species from sika deer and keds in Japan. Phylogenetic trees revealed Anaplasma sequences were grouped with the clusters of A. bovis and other ruminant-related Anaplasma, while Bartonella sequences were clustered with the novel Bartonella species lineages C, D, and E, which originated from Japan. Interestingly, a new independent lineage of novel Bartonella species was found in obtained specimens. We report the first molecular detection of Anaplasma and Bartonella on L. fortisetosa, which could represent infectious status of captive Eld’s deer in the zoo. Wild animals act as reservoirs for many pathogens, thus preventive measures in surrounding areas should be considered to prevent pathogen infection among animals or potential zoonotic infection among humans

Tick diversity and molecular detection of Anaplasma, Babesia, and Theileria from Khao Kheow open zoo, Chonburi Province, Thailand

Ticks are obligate blood-feeding ectoparasites notorious for their role as vectors for various pathogens, posing health risks to pets, livestock, wildlife, and humans. Wildlife also notably serves as reservoir hosts for tick-borne pathogens and plays a pivotal role in the maintenance and dissemination of these pathogenic agents within ecosystems. This study investigated the diversity of ticks and pathogens in wildlife and their habitat by examining ticks collected at Khao Kheow Open Zoo, Chonburi Province, Thailand. Tick samples were collected for 1 year from March 2021 to March 2022 by vegetation dragging and direct sampling from wildlife. A total of 10,436 ticks or 449 tick pools (1–50 ticks per pool) underwent screening for pathogen presence through conventional PCR and DNA sequencing. Out of the 298 samples (66.37%) where bacteria and protozoa were detected, encompassing 8,144 ticks at all stages, 114 positive samples from the PCR screenings were specifically chosen for detailed nucleotide sequencing and comprehensive analysis. Four species of ticks were conclusively identified through the application of PCR, namely, Rhipicephalus microplus, Dermacentor auratus, Haemaphysalis lagrangei, and Haemaphysalis wellingtoni. The highest infection rate recorded was for Anaplasma spp. at 55.23% (248/449), followed by Babesia spp. and Theileria spp. at 29.62% (133/449) and 16.26% (73/449), respectively. Among bacteria identified, three Anaplasma genotypes were closely related to an unidentified Anaplasma spp., A. phagocytophilum, and A. bovis. Among protozoa, only an unidentified Babesia spp. was found, whereas two Theileria genotypes found were closely related to unidentified Theileria spp. and T. equi. Significantly, our findings revealed coinfection with Anaplasma spp., Theileria spp., and Babesia spp. While blood samples from wildlife were not specifically collected to assess infection in this study, the data on the presence of various pathogens in ticks observed can serve as valuable indicators to assess the health status of wildlife populations and to monitor disease dynamics. The findings could be valuable in developing programs for the treatment, prevention, and control of tick-borne illnesses in this area. However, additional research is required to determine the ticks’ ability to transmit these pathogens and enhance the current understanding of the relationship among pathogens, ticks, and hosts

Evidence of Simultaneous Circulation of West Nile and Usutu Viruses in Mosquitoes Sampled in Emilia-Romagna Region (Italy) in 2009

BACKGROUND: In recent years human diseases due to mosquito-borne viruses were increasingly reported in Emilia-Romagna region (Italy), from the chikungunya virus in 2007 to the West Nile virus (WNV) in 2008. An extensive entomological survey was performed in 2009 to establish the presence and distribution of mosquito arboviruses in this region, with particular reference to flaviviruses. METHODOLOGY/PRINCIPAL FINDINGS: From May 6 to October 31, a total of 190,516 mosquitoes were sampled in georeferenced stations, grouped in 1,789 pools according date of collection, location, and species, and analyzed by reverse transcription polymerase chain reaction (RT-PCR) to detect the presence of RNA belong to Flavivirus genus. WNV was detected in 27 mosquito pools, producing sequences similar to those of birds and human strains obtained in 2008 outbreak, pointed out the probable virus overwintering. Isolation of WNV was achieved from one of these pools. Moreover 56 pools of mosquitoes tested positive for Usutu virus (USUV). Most PCR positive pools consisted of Culex pipiens, which also was the most analyzed mosquito species (81.4% of specimens); interestingly, USUV RNA was also found in two Aedes albopictus mosquito pools. Simultaneous circulation of WNV and USUV in the survey area was highlighted by occurrence of 8 mosquito WNV- and USUV-positive pools and by the overlaying of the viruses "hot spots", obtained by kernel density estimation (KDE) analysis. Land use of sampled stations pointed out a higher proportion of WNV-positive Cx. pipiens pool in rural environments respect the provenience of total sampled pool, while the USUV-positive pools were uniformly captured in the different environments. CONCLUSIONS/SIGNIFICANCE: Obtained data highlighting the possible role of Cx. pipiens mosquito as the main vector for WNV and USUV in Northern Italy, and the possible involvement of Ae. albopictus mosquito in USUV cycle. The described mosquito-based surveillance could constitute the foundation for a public health alert system targeting mosquito borne arboviruses

Vector competence of Ochlerotatus trivittatus (Coquillett), Aedes albopictus (Skuse), and Culex pipiens (L) (Diptera: Culicidae) for West Nile virus

West Nile virus (WNV) was recognized in North America in 1999. Avian species are the primary reservoir of the virus and can develop viremias with titers higher than 1010.0 CID50s/ml. Culex species of mosquitoes are the primary amplifying vectors. Many species of mammals are also susceptible to WNV but develop viremias that seldom exceed 105.0 CID50s/ml. Mammals may play a significant role in WNV ecology if mosquitoes that feed on them during periods of viremia can be infected and transmit the virus. The primary objective of this study was to determine vector competency of Ochlerotatus trivittatus (Coq.) a potential bridge vector that feeds primarily on mammals including humans and occasionally on birds. Susceptibility of Oc. trivittatus to WNV was compared to that of Aedes albopictus (Skuse) a known bridge vector, and Culex pipiens (L.), a primary amplifying vector by determining infection rates after feeding mosquitoes on chicks with blood meal titers (BMTs) of 102.5 to 1010.0 CID50s/ml. The ability of Oc. trivittatus to transmit WNV was determined by comparing transmission rates (%) of Oc. trivittatus, Ae. albopictus and Cx. pipiens by the capillary tube method following infection by blood meals with titers ranging from 102.5 to 109.5 CID50s/ml. Susceptibility of Oc. trivittatus and Cx. pipiens to WNV was essentially the same but greater than Ae. albopictus. The lowest infective BMTs for Oc. trivittatus, Ae. albopictus and Cx. pipiens were 104.5 , 105.5, and 104.5 CID50s/ml. The 50% infective BMTs for the 3 species were 106.0 10 6.6, and 106.2 CID50s/ml. Transmission rates of Oc. trivittatus, Ae. albopictus, and Cx. pipiens after blood meals with titers higher than 107.0 CID 50s/ml were 41.7, 72.4 and 46.8%. The lowest BMTs that resulted in transmission by the 3 species were 105.5, 107.0, and 10 5.5 CID50s/ml. These observations suggest that Oc. trivittatus might play a more significant role than Ae. albopictus in maintaining WNV in populations of mammals which typically develop low levels of viremia.</p

34-kDa salivary protein enhances duck Tembusu virus infectivity in the salivary glands of Aedes albopictus by modulating the innate immune response

Abstract Duck Tembusu virus (DTMUV) is an important flavivirus that can be transmitted to poultry via Aedes albopictus bites. Furthermore, humans residing in the DTMUV epidemic area display activated antiviral immune responses to local DTMUV isolates during the pathogenic invasion, thereby raising the primary concern that this flavivirus may be transmitted to humans via mosquito bites. Therefore, we identified the gene AALF004421, which is a homolog of the 34-kDa salivary protein (34 kDa) of Ae. albopictus and studied the salivary protein-mediated enhancement of DTMUV infection in Ae. albopictus salivary glands. We observed that double-stranded RNA-mediated silencing of the 34 kDa in mosquito salivary glands demonstrated that the silenced 34 kDa impaired DTMUV infectivity, similar to inhibition through serine protease. This impairment occurred as a consequence of triggering the innate immune response function of a macroglobulin complement-related factor (MCR). 34-kDa in the salivary gland which had similar activity as a serine protease, results in the abrogation of antimicrobial peptides production and strong enhance DTMUV replication and transmission. Although the function of the 34 kDa in Ae. albopictus is currently unknown; in the present study, we showed that it may have a major role in DTMUV infection in mosquito salivary glands through the suppression of the antiviral immune response in the earliest stages of infection. This finding provides the first identification of a prominently expressed 34 kDa protein in Ae. albopictus saliva that could serve as a target for controlling DTMUV replication in mosquito vectors

Molecular Analysis of Canine Filaria and Its Wolbachia Endosymbionts in Domestic Dogs Collected from Two Animal University Hospitals in Bangkok Metropolitan Region, Thailand

Canine filariasis is caused by several nematode species, such as Dirofilaria immitis, Dirofilaria repens, Brugia pahangi, Brugia malayi, and Acanthocheilonema reconditum. Zoonotic filariasis is one of the world&rsquo;s neglected tropical diseases. Since 2000, the World Health Organization (WHO) has promoted a global filarial eradication program to eliminate filariasis by 2020. Apart from vector control strategies, the infection control of reservoir hosts is necessary for more effective filariasis control. In addition, many studies have reported that Wolbachia is necessary for the development, reproduction, and survival of the filarial nematode. Consequently, the use of antibiotics to kill Wolbachia in nematodes has now become an alternative strategy to control filariasis. Previously, a case of subconjunctival dirofilariasis caused by Dirofilaria spp. has been reported in a woman who resides in the center of Bangkok, Thailand. Therefore, our study aimed to principally demonstrate the presence of filarial nematodes and Wolbachia bacteria in blood collected from domestic dogs from the Bangkok Metropolitan Region, Thailand. A total of 57 blood samples from dogs with suspected dirofilariasis who had visited veterinary clinics in Bangkok were collected. The investigations for the presence of microfilaria were carried out by using both microscopic and molecular examinations. PCR was used as the molecular detection method for the filarial nematodes based on the COI and ITS1 regions. The demonstration of Wolbachia was performed using PCR to amplify the FtsZ gene. All positive samples by PCR were then cloned and sequenced. The results showed that the filarial nematodes were detected in 16 samples (28.07%) using microscopic examinations. The molecular detection of filarial species using COI-PCR revealed that 50 samples (87.72%) were positive; these consisted of 33 (57.89%), 13 (22.81%), and 4 (7.02%) samples for D. immitis, B. pahangi, and B. malayi, respectively. While the ITS1-PCR showed that 41 samples (71.93%) were positive&mdash;30 samples (52.63%) were identified as containing D. immitis and 11 samples (19.30%) were identified to have B. pahangi, whereas B. malayi was not detected. Forty-seven samples (82.45%) were positive for Wolbachia DNA and the phylogenetic tree of all positive Wolbachia was classified into the supergroup C clade. This study has established fundamental data on filariasis associated with Wolbachia infection in domestic dogs in the Bangkok Metropolitan Region. An extensive survey of dog blood samples would provide valuable epidemiologic data on potential zoonotic filariasis in Thailand. In addition, this information could be used for the future development of more effective prevention and control strategies for canine filariasis in Thailand

Susceptibility of Fox Squirrels (Sciurus niger) to West Nile Virus by Oral Exposure

Fox squirrels (Sciurus niger) (five of eight) were infected with West Nile virus (WNV) when challenged by the oral route with 102.3 or 103.4 plaque forming units (PFU). The mean maximum serum WNV titer of infected fox squirrels was 105.1 PFU/mL and ranged from 104.6 to 105.6 PFU/mL. These levels of viremia are infectious for several mosquito vectors of WNV. This virus was also isolated from swabs of the oral and rectal cavities, and urine swabs between day 5 and 9 postexposure (p.e.) in amounts as high as 102.0, 102.8, and 102 PFU, respectively. WNV RNA was detected in salivary gland and/or kidney tissue of three squirrels between day 65 and 72 p.e. in the presence of WNV neutralizing antibody, suggesting that long-term persistent infection occurs in fox squirrels. These observations justify further studies to determine if nonarthropod transmission and long-term persistent infection occur naturally in fox squirrels and contribute to trans-seasonal maintenance of WNV.This article is from Vector-Borne and Zoonotic Diseases 10 (2009): 207, 10.1089/vbz.2008.0158. Posted with permission.</p