Metagenomic analysis of Aedes aegypti and Culex quinquefasciatus mosquitoes from Grenada, West Indies.

The mosquitoes Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) and Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) are two major vectors of arthropod-borne pathogens in Grenada, West Indies. As conventional vector control methods present many challenges, alternatives are urgently needed. Manipulation of mosquito microbiota is emerging as a field for the development of vector control strategies. Critical to this vector control approach is knowledge of the microbiota of these mosquitoes and finding candidate microorganisms that are common to the vectors with properties that could be used in microbiota modification studies. Results showed that bacteria genera including Asaia, Escherichia, Pantoea, Pseudomonas, and Serratia are common to both major arboviral vectors in Grenada and have previously been shown to be good candidates for transgenetic studies. Also, for the first time, the presence of Grenada mosquito rhabdovirus 1 is reported in C. quinquefasciatus.

High prevalence of Phasi Charoen-like virus from wild-caught Aedes aegypti in Grenada, W.I. as revealed by metagenomic analysis.

Arboviruses cause diseases of significant global health concerns. Interactions between mosquitoes and their microbiota as well as the important role of this interaction in the mosquito's capacity to harbor and transmit pathogens have emerged as important fields of research. Aedes aegypti is one of the most abundant mosquitoes in many geographic locations, a vector capable of transmitting a number of arboviruses such as dengue and Zika. Currently, there are few studies on the metavirome of this mosquito particularly in the Americas. This study analyzes the metavirome of A. aegypti from Grenada, a Caribbean nation with tropical weather, abundant A. aegypti, and both endemic and arboviral pathogens transmitted by this mosquito. Between January and December 2018, 1152 mosquitoes were collected from six semi-rural locations near houses in St. George Parish, Grenada, by weekly trapping using BG-Sentinel traps. From these, 300 A. aegypti were selected for analysis. The metavirome was analyzed using the Illumina HiSeq 1500 for deep sequencing. The generation sequencing library construction protocol used was NuGEN Universal RNA with an average read length of 125 bp. Reads were mapped to the A. aegypti assembly. Non-mosquito reads were analyzed using the tools FastViromeExplorer. The NCBI total virus, RNA virus, and eukaryotic virus databases were used as references. The metagenomic comparison analysis showed that the most abundant virus-related reads among all databases and assemblies was Phasi Charoen-like virus. The Phasi Charoen-like virus results are in agreement to other studies in America, Asia and Australia. Further studies using wild-caught mosquitoes is needed to assess the impact of this insect-specific virus on the A. aegypti lifecycle and vector capacity.

Phylogenetic relationships and diversity of bat-associated Leptospira and the histopathological evaluation of these infections in bats from Grenada, West Indies.

Bats can harbor zoonotic pathogens, but their status as reservoir hosts for Leptospira bacteria is unclear. During 2015-2017, kidneys from 47 of 173 bats captured in Grenada, West Indies, tested PCR-positive for Leptospira. Sequence analysis of the Leptospira rpoB gene from 31 of the positive samples showed 87-91% similarity to known Leptospira species. Pairwise and phylogenetic analysis of sequences indicate that bats from Grenada harbor as many as eight undescribed Leptospira genotypes that are most similar to known pathogenic Leptospira, including known zoonotic serovars. Warthin-Starry staining revealed leptospiral organisms colonizing the renal tubules in 70% of the PCR-positive bats examined. Mild inflammatory lesions in liver and kidney observed in some bats were not significantly correlated with renal Leptospira PCR-positivity. Our findings suggest that Grenada bats are asymptomatically infected with novel and diverse Leptospira genotypes phylogenetically related to known pathogenic strains, supporting the hypothesis that bats may be reservoirs for zoonotic Leptospira.

Initial development and preliminary evaluation of a multiplex bead assay to detect antibodies to Ehrlichia canis, Anaplasma platys, and Ehrlichia chaffeensis outer membrane peptides in naturally infected dogs from Grenada, West Indies.

Tick-borne bacteria, Ehrlichia canis, Anaplasma platys, and Ehrlichia chaffeensis are significant pathogens of dogs worldwide, and coinfections of E. canis and A. platys are common in dogs on the Caribbean islands. We developed and evaluated the performance of a multiplex bead-based assay to detect antibodies to E. canis, A. platys, and E. chaffeensis peptides in dogs from Grenada, West Indies, where E. canis and A. platys infections are endemic. Peptides from outer membrane proteins of P30 of E. canis, OMP-1X of A. platys, and P28-19/P28-14 of E. chaffeensis were coupled to magnetic beads. The multiplex peptide assay detected antibodies in dogs experimentally infected with E. canis and E. chaffeensis, but not in an A. platys experimentally infected dog. In contrast, the multiplex assay and an in-house enzyme-linked immunosorbent assay (ELISA) detected A. platys antibodies in naturally infected Grenadian dogs. Following testing of 104 Grenadian canine samples, multiplex assay results had good agreement with commercially available ELISA and immunofluorescent assay for E. canis antibody-positive dogs ( K values of 0.73 and 0.84), whereas A. platys multiplex results had poor agreement with these commercial assays ( K values of -0.02 and 0.01). Prevalence of seropositive E. canis and A. platys Grenadian dogs detected by the multiplex and commercial antibody assays were similar to previous reports. Although the multiplex peptide assay performed well in detecting the seropositive status of dogs to E. canis and had good agreement with commercial assays, better antigen targets are necessary for the antibody detection of A. platys.

Genotypes, Antibiotic Resistance, and ST-8 Genetic Clone in Campylobacter Isolates from Sheep and Goats in Grenada.

Rectal swabs from 155 sheep and 252 goats from Grenada were evaluated to determine the prevalence of Campylobacter spp., antibiotic resistance, and multilocus sequence types. Fifteen Campylobacter isolates were obtained (14 C. jejuni and 1 C. coli). The prevalence (3.7%) did not differ significantly between sheep (4.5%) and goats (3.2%). Among the seven antimicrobials tested, resistance was only detected for tetracycline (30.8%) and metronidazole (38.5%). Campylobacter isolates showed no significant difference between sheep and goats for type of antimicrobial resistance or percent of resistant isolates. Twelve of the isolates were successfully genotyped consisting of four recognized clonal complexes and three novel sequence types. Importantly, one isolate from one goat was identified as the C. jejuni sequence type-8, a zoonotic and tetracycline-resistant clone reported to be a highly virulent clone associated with ovine abortion in the USA. Although most samples were from comingled sheep and goat production units, there were no shared sequence types between these two host species. None of the sequence types identified in this study have previously been reported in poultry in Grenada, suggesting sheep- and goat-specific Campylobacter clones in Grenada. This is the first report of genotyping of Campylobacter isolates from sheep and goats in the Eastern Caribbean.

Doxycycline treatment of asymptomatic dogs seropositive for Ehrlichia canis

In Grenada, West Indies dogs are at frequent exposure to the rickettsial pathogen, Ehrlichia canis, as demonstrated by high seroprevalence rates. However, many of these seropositive dogs are clinically normal. In this study we identified clinically normal, E. canis seropositive dogs and assigned half to an antibiotic treatment group and half to a no treatment group. All dogs were evaluated for the presence of E. canis DNA by PCR on whole blood before, during and after treatment. Only one seropositive dog was also PCR+ before treatment. Our results suggest that most clinically normal, E. canis seropositive dogs in a highly endemic geographic area are not concurrently infected and thus routine treatment of clinically normal, seropositive dogs is not warranted.

Translocation of the B cell receptor to lipid rafts is inhibited in B cells from BLV-infected, persistent lymphocytosis cattle.

Bovine leukemia virus (BLV) infection causes a significant polyclonal expansion of CD5(+), IgM+ B lymphocytes known as persistent lymphocytosis (PL) in approximately 30% of infected cattle. There is evidence that this expanded B cell population has altered signaling, and resistance to apoptosis has been proposed as one mechanism of B cell expansion. In human and murine B cells, antigen binding initiates movement of the B cell receptor (BCR) into membrane microdomains enriched in sphingolipids and cholesterol, termed lipid rafts. Lipid rafts include members of the Src-family kinases and exclude certain phosphatases. Inclusion of the BCR into lipid rafts plays an important role in regulation of early signaling events and subsequent antigen internalization. Viral proteins may also influence signaling events in lipid rafts. Here we demonstrate that the largely CD5(+) B cell population in PL cattle has different mobilization and internalization of the BCR when compared to the largely CD5-negative B cells in BLV-negative cattle. Unlike B cells from BLV-negative cattle, the BCR in B cells of BLV-infected, PL cattle resists movement into lipid rafts upon stimulation and is only weakly internalized. Expression of viral proteins as determined by detection of the BLV transmembrane (TM) envelope glycoprotein gp30 did not alter these events in cells from PL cattle. This exclusion of the BCR from lipid rafts may, in part, explain signaling differences seen between B cells of BLV-infected, PL, and BLV-negative cattle and the resistance to apoptosis speculated to contribute to persistent lymphocytosis.

Rhodococcus equi secreted antigens are immunogenic and stimulate a type 1 recall response in the lungs of horses immune to R. equi infection.

Rhodococcus equi is an opportunistic pathogen in immunocompromised humans and an important primary pathogen in young horses. Although R. equi infection can produce life-threatening pyogranulomatous pneumonia, most foals develop a protective immune response that lasts throughout life. The antigen targets of this protective response are currently unknown; however, Mycobacterium tuberculosis is a closely related intracellular pathogen and provides a model system. Based on previous studies of M. tuberculosis protective antigens released into culture filtrate supernatant (CFS), a bacterial growth system was developed for obtaining R. equi CFS antigens. Potential immunogens for prevention of equine rhodococcal pneumonia were identified by using immunoblots. The 48-h CFS contained five virulence-associated protein bands that migrated between 12 and 24 kDa and were recognized by sera from R. equi-infected foals and immune adult horses. Notably, the CFS contained the previously characterized proteins VapC, VapD, and VapE, which are encoded by genes on the R. equi virulence plasmid. R. equi CFS was also examined for the ability to stimulate a type 1-like memory response in immune horses. Three adult horses were challenged with virulent R. equi, and cells from the bronchoalveolar lavage fluid were recovered before and 1 week after challenge. In vitro stimulation of pulmonary T-lymphocytes with R. equi CFS resulted in significant proliferation and a significant increase in gamma interferon mRNA expression 1 week after challenge. These results were consistent with a memory effector response in immune adult horses and provide evidence that R. equi CFS proteins are antigen targets in the immunoprotective response against R. equi infection.

Clearance of virulent but not avirulent Rhodococcus equi from the lungs of adult horses is associated with intracytoplasmic gamma interferon production by CD4+ and CD8+ T lymphocytes.

Rhodococcus equi is a gram-positive bacterium that infects alveolar macrophages and causes rhodococcal pneumonia in horses and humans. The virulence plasmid of R. equi appears to be required for both pathogenicity in the horse and the induction of protective immunity. An understanding of the mechanisms by which virulent R. equi circumvents protective host responses and by which bacteria are ultimately cleared is important for development of an effective vaccine. Six adult horses were challenged with either virulent R. equi or an avirulent, plasmid-cured derivative. By using a flow cytometric method for intracytoplasmic detection of gamma interferon (IFN-gamma) in equine bronchoalveolar lavage fluid (BALF) cells, clearance of the virulent strain was shown to be associated with increased numbers of pulmonary CD4(+) and CD8(+) T lymphocytes producing IFN-gamma. There was no change in IFN-gamma-positive cells in peripheral blood, suggesting that a type 1 recall response at the site of challenge was protective. The plasmid-cured strain of R. equi was cleared in horses without a significant increase in IFN-gamma-producing T lymphocytes in BALF. In contrast to these data, a previous report in foals suggested an immunomodulating role for R. equi virulence plasmid-encoded products in downregulating IFN-gamma expression by equine CD4(+) T lymphocytes. Intracytoplasmic detection of IFN-gamma provides a method to better determine whether modulation of macrophage-activating cytokines by virulent strains occurs uniquely in neonates and contributes to their susceptibility to rhodococcal pneumonia.

Antiviral activity of shiga toxin requires enzymatic activity and is associated with increased permeability of the target cells.

This study expanded our earlier finding that Shiga toxin type 1 (Stx1) has activity against bovine leukemia virus (BLV) (W. A. Ferens and C. J. Hovde, Infect. Immun. 68:4462-4469, 2000). The Stx molecular motifs required for antiviral activity were identified, and a mechanism of Stx action on virally infected cells is suggested. Using inhibition of BLV-dependent spontaneous lymphocyte proliferation as a measure of antiviral activity, we showed that Stx2 had antiviral activity similar to that of Stx1. Enzymatic and antiviral activities of three StxA1 chain mutants deficient in enzymatic activity or aspects of receptor-mediated cytotoxicity were compared. Using protein synthesis inhibition to measure enzymatic activity, the mutant E167D was 300-fold less catalytically active than wild-type StxA1, was minimally active in antiviral assays, and did not inhibit synthesis of viral proteins. Two StxA1 mutants, A231D-G234E and StxA(1)1 (enzymatically active but unable to kill cells via the classical receptor-mediated route), had undiminished antiviral activity. Although binding of radiolabeled StxA1 to bovine blood cells or to free virus was not detected, flow cytometric analysis showed that the number of BLV-expressing cells were specifically reduced in cultures treated with Stx. These unique and rare lymphocytes were highly permeable to 40- and 70-kDa fluorescent dextrans, indicating that direct absorption of toxins by virus-expressing cells is a potential mechanism of target cell intoxication. These results support the hypothesis that Stx-producing Escherichia coli colonization of the gastrointestinal tract may benefit ruminant hosts by the ability of Stxs to exert antiviral activity.

Bovine leukemia virus gp30 transmembrane (TM) protein is not tyrosine phosphorylated: examining potential interactions with host tyrosine-mediated signaling.

Bovine leukemia virus (BLV) causes persistent lymphocytosis, a preneoplastic, polyclonal expansion of B lymphocytes. The expansion increases viral transmission to new hosts, but the mechanisms of this expansion have not been determined. We hypothesized that BLV infection contributes to B-cell expansion by signaling initiated via viral transmembrane protein motifs undergoing tyrosine phosphorylation. Viral mimicry of host cell proteins is a well-demonstrated mechanism by which viruses may increase propagation or decrease recognition by the host immune system. The cytoplasmic tail of BLV transmembrane protein gp30 (TM) has multiple areas of homology to motifs of host cell signaling proteins, including two immunoreceptor tyrosine-based activation motifs (ITAMs) and two immunoreceptor tyrosine-based inhibition motifs (ITIMs), which are homologous to B-cell receptor and inhibitory co-receptor motifs. Signaling by these motifs in B cells typically relies on tyrosine phosphorylation, followed by interactions with Src-homology-2 (SH2) domains of nonreceptor protein tyrosine kinases or phosphatases. Phosphorylation of tyrosine residues in the cytoplasmic tail of TM was tested in four systems including ex vivo cultured peripheral blood mononuclear cells from BLV infected cows, BLV-expressing fetal lamb kidney cell and bat lung cell lines, and DT40 B cells transfected with a fusion of mouse extracellular CD8alpha and cytoplasmic TM. No phosphorylation of TM was detected in our experiments in any of the cell types utilized, or with various stimulation methods. Detection was attempted by immunoblotting for phosphotyrosines, or by metabolic labeling of cells. Thus BLV TM is not likely to modify host signal pathways through interactions between phosphorylated tyrosines of the ITAM or ITIM motifs and host-cell tyrosine kinases or phosphatases.