Comparison of surveillance trapping methods to monitor Culicoides biting midge activity in Trinidad, West Indies.

Culicoides biting midges (Diptera: Ceratopogonidae) are biting nuisances and arbovirus vectors of both public health and veterinary significance in Trinidad. We compared sampling methods to define the behaviour and bionomics of adult Culicoides populations at a commercial dairy goat farm. Three static trap designs were compared: (a) Centre for Disease Control (CDC) downdraft UV trap; (b) CDC trap with an incandescent bulb and (c) CDC trap with semiochemical lure consisting of R-(-)-1-octen-3-ol and CO2 (no bulb). Sweep netting was used to define diel periodicity. A total of 30,701 biting midges were collected using static traps, dominated by female Culicoides furens (>70% of trap collections across all three designs). There was no significant difference in the Margalef's index between the three traps; however, trap designs A and C collected a significantly greater number of individuals than trap B, and trap C gained highest species richness. The greatest species richness and abundance of Culicoides collected by sweep net was observed between 6:00 and 6:15 pm and notable differences in the crepuscular activity pattern of several species were identified. Comparative data on Culicoides species richness, abundance, sex and reproductive status is discussed and can be used to improve surveillance strategies, research designs and risk management.

Seroprevalence of economically important viral pathogens in swine populations of Trinidad and Tobago, West Indies.

The objective of this study was to evaluate the seroprevalence and identify the strains of swine influenza virus (SwIV), as well as the seroprevalence of porcine parvovirus (PPV), transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV-2), and classical swine fever virus (CSFV) in pigs in Trinidad and Tobago (T&T). Blood samples (309) were randomly collected from pigs at farms throughout T&T. Serum samples were tested for the presence of antibodies to the aforementioned viruses using commercial ELISA kits, and the circulating strains of SwIV were identified by the hemagglutination inhibition test (HIT). Antibodies against SwIV were detected in 114 out of the 309 samples (37%). Out of a total of 26 farms, 14 tested positive for SwIV antibodies. HI testing revealed high titers against the A/sw/Minnesota/593/99 H3N2 strain and the pH1N1 2009 pandemic strain. Antibodies against PPV were detected in 87 out of the 309 samples (28%), with 11 out of 26 farms testing positive for PPV antibodies. Antibodies against PCV-2 were detected in 205 out of the 309 samples tested (66%), with 25 out of the 26 farms testing positive for PCV-2 antibodies. No antibodies were detected in any of the tested pigs to PRRSV, TGEV, PRCV, or CSFV.

Virome of Ixodes ricinus, Dermacentor reticulatus, and Haemaphysalis concinna Ticks from Croatia.

Tick-borne diseases are a serious threat to both public and veterinary health. In this study, we used high-throughput sequencing to characterize the virome of three tick species implicated in the spread of vector-borne disease throughout Croatia. Ten viruses were identified, including seven potential novel species within the viral families Flaviviridae, Nyamiviridae, Rhabdoviridae, Peribunyaviridae, Phenuiviridae, and Nairoviridae.

An international, interlaboratory ring trial confirms the feasibility of an extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

Novel quaranjavirus and other viral sequences identified from ticks parasitizing hunted wildlife in Trinidad and Tobago.

Hunters are at a higher risk for exposure to zoonotic pathogens due to their close interactions with wildlife and arthropod vectors. In this study, high throughput sequencing was used to explore the viromes of two tick species, Amblyomma dissimile and Haemaphysalis juxtakochi, removed from hunted wildlife in Trinidad and Tobago. We identified sequences from 3 new viral species, from the viral families Orthomyxoviridae, Chuviridae and Tetraviridae in A. dissimile.

An international, interlaboratory ring trial confirms the feasibility of an open-source, extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is an open-access qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that open-access, direct RT-PCR assays are a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

Characterization of novel, pathogenic field strains of infectious bronchitis virus (IBV) in poultry in Trinidad and Tobago.

Avian coronaviruses, including infectious bronchitis virus (IBV) and turkey coronavirus (TCoV), are economically important viruses affecting poultry worldwide. IBV is responsible for causing severe losses to the commercial poultry sector globally. The objectives of this study were to identify the viruses that were causing outbreaks of severe respiratory disease in chickens in Trinidad and Tobago (T&T) and to characterize the strains. Swab samples were collected from birds showing severe respiratory signs in five farms on the island of Trinidad. Samples were tested for the presence of IBV, as well as avian influenza virus (AIV), Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) by real-time reverse transcription polymerase chain reaction (qRT-PCR). All samples from the five farms tested negative for AIV, NDV and aMPV; however, samples from clinically affected birds in all five of the farms tested positive for IBV. Genetic data revealed the presence of TCoV in chickens on two of the farms. Interestingly, these two farms had never reared turkeys. Phylogenetic analysis showed that IBV S1 sequences formed two distinct clusters. Two sequences grouped with vaccine strains within the GI-1 lineage, whereas three sequences grouped together, but separately from other defined lineages, forming a likely new lineage of IBV. Pairwise comparison revealed that the three unique variant strains within the distinct lineage of IBV were significantly different in their S1 nucleotide coding regions from viruses in the closest lineage (16% difference) and locally used vaccine strains (>20% difference). Results also suggested that one of the samples was a recombinant virus, generated from a recombination event between a Trinidad virus of the GI-1 lineage and a Trinidad virus of the newly defined lineage. Many amino acid differences were also observed between the S1 coding regions of the circulating field and vaccine strains, indicating that the IBV vaccines may not be protective. Vaccine-challenge studies are however needed to prove this.

The Serological Prevalence of Rabies Virus-Neutralizing Antibodies in the Bat Population on the Caribbean Island of Trinidad.

: Rabies virus (RABV) is the only lyssavirus known to be present within the Caribbean. The island of Trinidad, is richly diverse in chiropteran fauna and endemic for bat-transmitted rabies with low RABV isolation rates observed in this population. We aimed to determine the seroprevalence of rabies virus neutralizing antibodies (RVNA) in light of spatio-temporal and bat demographic factors to infer the extent of natural exposure to RABV in the Trinidadian bat population. RVNA titers were determined by the RABV micro-neutralization test on 383 bat samples representing 21 species, comprising 30.9% of local bat diversity, from 31 locations across the island over 5 years. RVNA was positively detected in 33 samples (8.6%) representing 6 bat species (mainly frugivorous) with titers ranging from 0.1 to 19 IU/mL (mean 1.66 IU/mL). The analyses based on a multivariable binomial generalised linear mixed-effects model showed that bat age and year of capture were significant predictors of seropositivity. Thus, juvenile bats were more likely to be seropositive when compared to adults (estimate 1.13; p = 0.04) which may suggest early exposure to the RABV with possible implications for viral amplification in this population. Temporal variation in rabies seropositivity, 2012-2014 versus 2015-2017 (estimate 1.07; p = 0.03) may have been related to the prevailing rabies epizootic situation. Regarding other factors investigated, RVNA was found in bats from both rural and non-rural areas, as well as in both hematophagous and non-hematophagous bat species. The most common seropositive species, Artibeusjamaicensisplanirostris is ubiquitous throughout the island which may potentially facilitate human exposure. The findings of this study should be factored into public health assessments on the potential for rabies transmission by non-hematophagous bats in Trinidad.

Identification of four serotypes of fowl adenovirus in clinically affected commercial poultry co-infected with chicken infectious anaemia virus in Trinidad and Tobago.

Fowl adenovirus (FAdV), which causes the high-impact diseases such as inclusion body hepatitis and hepatitis-hydropericardium syndrome, is of major concern to the poultry industry internationally. This study was carried out in direct response to mortality rates of up to 75% in commercial broiler flocks in Trinidad, West Indies. Symptoms in 3- to 8-week-old broilers and 13- to 18-week-old pullets pointed to infection with an immunosuppressive viral pathogen. The objectives of the study were to determine whether the infectious agent FAdV, along with other viral pathogens, was responsible for the clinical disease, and to obtain information on the serotypes of FAdV that were infecting the birds. Tissue samples from clinically affected birds from eight different farms were tested for chicken infectious anaemia virus (CIAV) and infectious bursal disease virus (IBDV) by real-time reverse transcription polymerase chain reaction (PCR) and for FAdV by conventional PCR. The birds tested positive for FAdV and CIAV, but negative for IBDV. The gene corresponding to the L1 loop of the hexon protein for FAdV was amplified and sequenced. Phylogenetic analysis of seven FAdV strains inferred that four serotypes were likely to be circulating in the chickens. Well supported genetic relatedness was observed for serotype 8a (97.8%), 8b (97.8%), 9 (95.8%) and 11 (98.8%-99.5%). This is the first published report from Trinidad and Tobago on the presence and circulation of pathogenic FAdV strains, in combination with CIAV, in poultry. The data demonstrate a possible need for the introduction of serotype-specific vaccines against FAdV, as well as vaccines against CIAV, in broilers in the region and emphasize the importance of maintaining high levels of biosecurity on farms to prevent the spread of these potentially devastating viruses between farms.

Detection of a novel reassortant epizootic hemorrhagic disease virus serotype 6 in cattle in Trinidad, West Indies, containing nine RNA segments derived from exotic EHDV strains with an Australian origin.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted orbivirus that infects domestic and wild ruminants in many parts of the world. Of the eight proposed serotypes, only EHDV-1, 2 and 6 have been reported to be present in the Americas. Following the identification of a virulent EHD-6 reasssortant virus in the USA in 2007 (EHDV-6 Indiana), with outer coat protein segments derived from an Australian strain of EHDV and all remaining segments derived from a locally circulating EHDV-2 strain, questions have remained about the origin of the Australian parent strain and how it may have arrived in the USA. When EHDV-6 was identified in asymptomatic cattle imported into the Caribbean island of Trinidad in 2013, full genome sequencing was carried out to further characterise the virus. The EHDV-6 Trinidad was a reassortant virus, with 8 of its 10 segments, being derived from the same exotic Australian EHDV-6 strain as the VP2 and VP5 present in the EHDV-6 Indiana strain from the USA. Analyses of the two remaining segments revealed that segment 8 showed the highest nucleotide identity (90.4%) with a USA New Jersey strain of EHDV-1, whereas segment 4 had the highest nucleotide identity (96.5%) with an Australian EHDV-2 strain. This data strongly suggests that the Trinidad EHDV-6 has an Australian origin, receiving its segment 4 from a reassortment event with an EHDV-2 also from Australia. This reassortant virus likely came to the Americas, where it received its segment 8 from a locally-circulating (as yet unknown) EHDV strain. This virus then may have gained entry into the USA, where it further reassorted with a known locally-circulating EHDV-2, the resulting strain being EHDV-6 Indiana. This study therefore identifies, for the first time, the likely minor parent virus of the EHDV-6 currently circulating in the USA.

Identification and characterization of epizootic hemorrhagic disease virus serotype 6 in cattle co-infected with bluetongue virus in Trinidad, West Indies.

Epizootic hemorrhagic disease virus (EHDV) is an economically important virus that can cause severe clinical disease in deer and to a lesser extent cattle. This study set out to determine and characterize which EHDV serotypes were circulating in Trinidad. Serum and whole blood samples were collected monthly for six months from a cohort of cattle imported to Trinidad from the USA. Results revealed that all the cattle seroconverted to EHDV within six months of their arrival, with EHDV RNA being detected in the samples just prior to antibodies, as expected. Serotyping assays revealed that a single serotype (EHDV-6) was circulating in the cattle. Sequencing of the surface viral protein (VP2) of EHDV-6, followed by phylogenetic analysis, revealed that the Trinidad EHDV-6 strain was closely related to EHDV-6 viruses found in Guadeloupe (2010), Martinique (2010) and USA (2006), with 96-97.2% nucleotide identity. The Trinidad EHDV-6 VP-2 shared 97.2% identity with the Australian EHDV-6 prototype strain, classifying it within the eastern topotype clade. Bayesian coalescent analysis support Australia as the most probable source for the EHDV-6 VP2 sequences in the Americas and Caribbean region and suggests that the they diverged from the Australian prototype strain around 1966 (95% HPD 1941-1979).

Serological evidence for eight globally important poultry viruses in Trinidad & Tobago.

Viruses affecting poultry cause significant levels of disease leading to severe economic losses among poultry farmers worldwide. The Americas region continues to be vulnerable to the spread of poultry viruses across the continents and Caribbean island chains. In Trinidad and Tobago (T&T) there is limited information on the viruses circulating in poultry. Many flock are vulnerable to infection and there are occasional outbreaks of disease resulting in high levels of morbidity and mortality. This study aims to identify important viruses of poultry circulating in T&T through a broad-based surveillance approach. Serum samples from 29 layer farms in Trinidad and 14 layer farms in Tobago were collected from the eldest laying hens. Samples were tested from unvaccinated birds for antibodies by enzyme-linked immunosorbent assay (ELISA) against Avian influenza virus (AIV), Infectious bronchitis virus (IBV), Newcastle disease virus (NDV), Infectious laryngotracheitis virus (ILTV), Avian pneumovirus (APV), Infectious bursal disease virus (IBDV), Fowl adenovirus Gp1 (FADV) and Egg drop syndrome virus (EDSV). In Trinidad, the estimated true seroprevalence levels of antibodies were 0% (CI 95%: 0-0%) for AIV, 100% (CI 95%: 97-100%) for IBV, 79.8% (CI 95%: 70.6-86.9%) for NDV, 1% (CI 95%: 0-2.6%) for ILTV, 67.55% (CI 95%: 62.3-72.4%) for APV, 94.93% (CI 95%: 88.0-98.6%) for IBDV, 100% (CI 95%: 99.7-100%) for FADV and 67.8% (CI 95%: 62.4-72.8%) for EDSV. In Tobago, seroprevalence levels were 0% (CI 95%: 0-0%) for AIV, 100% (CI 95%: 95.6-100%) for IBV, 80.5% (CI 95%: 70.1-88.5%) for NDV, 29.9% (CI 95%: 20.8-40.6%) for ILTV, 100% (CI 95%: 97.7-100%) for APV, 97.1% (CI95%: 89.9-100%) for IBDV, 100% (CI 95%: 97.5-100%) for FADV and 100% (CI 95%: 99-100%) for EDSV. The results reveal strong evidence for the circulation of IBV, NDV, APV, IBDV, FADV and EDSV in layer poultry on both islands, as well as ILTV in Tobago.

Serosurvey for Infectious Agents Associated with Subfertility and Abortion in Dairy Cattle in Trinidad and Tobago, West Indies.

Despite frequent reports of subfertility and abortion in dairy cattle in Trinidad and Tobago (T&T), little is known about the potential infectious and non-infectious causes. This study set out to investigate possible infectious causes of reproductive problems by measuring the seroprevalence of four of the most significant reproductive pathogens in dairy cattle worldwide: Brucella abortus (B. abortus); Neospora caninum (N. caninum), Bovine Viral Diarrhoea virus (BVDV), and Infectious Bovine Rhinotracheitis virus (IBRV). These four reproductive pathogens have been suspected to be present in dairy cattle in T&T for some time but, previously, studies have not been carried out to confirm their presence. Bulk milk samples were collected from 92 dairy farms across Trinidad, representing a total of 1177 dairy cattle. Four dairy farms were selected for individual milk sampling to assess in-farm seroprevalence levels. Milk samples were tested for antibodies to the four pathogens by commercial ELISA kits. The overall farm seroprevalence was 62% for N. caninium and 23% for IBRV, and no antibodies were detected in any of the bulk milk samples for B. abortus or BVDV. Mixed infections for IBRV and N. caninum were common. Seroprevalence levels were between 8% and 65% for N. caninum and between 3% and 53% IBRV on the four individual farms. These results reveal the presence of IBRV and N. caninum for the first time on the island of Trinidad and importantly reveal no evidence for the circulation of BVDV or B. abortus in dairy cattle in Trinidad.

Evaluation of the safety, immunogenicity and efficacy of three capripoxvirus vaccine strains against lumpy skin disease virus.

The safety, immunogenicity and efficacy of three commercially available vaccines against lumpy skin disease (LSD) in cattle have been evaluated using a combination of vaccine challenge experiments and the monitoring of immune responses in vaccinated animals in the field. The three vaccines evaluated in the study included two locally produced (Ethiopian) vaccines (lumpy skin disease virus (LSDV) Neethling and Kenyan sheep and goat pox (KSGP) O-180 strain vaccines) and a Gorgan goat pox (GTP) vaccine manufactured by Jordan Bio-Industries Centre (JOVAC). The latter vaccine was evaluated for the first time in cattle against LSDV. The Ethiopian Neethling and KSGPO-180 vaccines failed to provide protection in cattle against LSDV, whereas the Gorgan GTP vaccine protected all the vaccinated calves from clinical signs of LSD. There was no significant difference in protective efficacy detected between two dosage levels (P=0.2, P=0.25, and P=0.1 for KSGP, Neethling and Gorgan vaccines, respectively). Additionally, the Gorgan GTP vaccinated cattle showed stronger levels of cellular immune responses measured using Delayed-Type Hypersensitivity (DTH) reactions at the vaccination site indicating higher levels of immunogenicity produced by the GTPV vaccine in cattle, as opposed to the other two vaccines. This study indicated, for the first time, that the Gorgan GTP vaccine can effectively protect cattle against LSDV and that the Neethling and KSGP O-180 vaccine were not protective. The results emphasise the need for molecular characterization of the Neethling and KSGP O-180 vaccine seed viruses used for vaccine production in Ethiopia. In addition, the potency and efficacy testing process of the Ethiopian LSD Neethling and KSGP O-180 vaccines should be re-evaluated.

Circulation of bluetongue virus in goats in the Karamoja region of Uganda.

The presence of bluetongue virus (BTV) in indigenous goats from the Karamoja region of northern Uganda was investigated. A total of 300 goats were sampled (serum and whole blood) from five districts within the Karamoja region. The samples were analysed for the presence of bluetongue (BT) antibodies using a commercial Enzyme-linked immunosorbent assay (ELISA) and for the presence of BTV viral RNA by real-time Reverse transcription polymerase chain reaction (RT-PCR), because BTV is an RNA virus. Of the 300 goats tested, 269 (90%) were positive for BTV antibodies, indicating high levels of BTV circulation within the region. Out of the 150 whole blood samples tested for the presence of the virus by real-time RT-PCR, 84 (56%) were positive for BTV RNA. This study, which is the first of its kind in Uganda, showed a high seroprevalence of BT antibodies and active circulation of BTV in a high proportion of goats in the Karamoja region.

Neutralising antibody responses in cattle and sheep following booster vaccination with two commercial inactivated bluetongue virus serotype 8 vaccines.

Cattle and sheep that had received a primary course of vaccination with an inactivated bluetongue virus serotype 8 (BTV-8) vaccine were booster vaccinated 6 or 12 months later with the homologous vaccine or an alternative inactivated BTV-8 vaccine and neutralising antibody responses were determined. Antibody titres to the alternative vaccine were significantly higher than to the homologous vaccine (P=0.013) in cattle. There was no significant difference between the antibody responses to alternative and homologous vaccines in sheep. These data indicate that cattle and sheep primed with one inactivated BTV-8 vaccine may be effectively boosted with an alternative commercial inactivated BTV-8 vaccine.

Protection of European domestic pigs from virulent African isolates of African swine fever virus by experimental immunisation.

African swine fever (ASF) is an acute haemorrhagic disease of domestic pigs for which there is currently no vaccine. We showed that experimental immunisation of pigs with the non-virulent OURT88/3 genotype I isolate from Portugal followed by the closely related virulent OURT88/1 genotype I isolate could confer protection against challenge with virulent isolates from Africa including the genotype I Benin 97/1 isolate and genotype X Uganda 1965 isolate. This immunisation strategy protected most pigs challenged with either Benin or Uganda from both disease and viraemia. Cross-protection was correlated with the ability of different ASFV isolates to stimulate immune lymphocytes from the OURT88/3 and OURT88/1 immunised pigs.