DNA vaccination of the American crow (Corvus brachyrhynchos) provides partial protection against lethal challenge with West Nile virus

The New York 1999 strain of West Nile virus (WNV) is nearly 100% fatal in the American crow (Corvus brachyrhynchos). We evaluated four WNV vaccine formulations in American crows, including intramuscular (i.m.) DNA vaccine, i.m. DNA vaccine with adjuvant, orally administered microencapsulated DNA vaccine, and i.m. killed vaccine. Neutralizing antibodies developed in approximately 80% of crows that received the DNA vaccine i.m. (with or without adjuvant), and in 44% that received the killed vaccine. However, no crows that received the oral microencapsulated DNA vaccine or the placebo developed WNV antibodies. All crows were challenged 10 wk after initial vaccination. No unvaccinated crows survived challenge, and survival rates were 44% (i.m. DNA vaccine), 60% (i.m. DNA vaccine with adjuvant), 0% (oral microencapsulated DNA vaccine), and 11% (killed vaccine). Peak viremia titers in the birds that survived were significantly lower as compared to titers in birds that died. Parenteral administration of a WNV DNA vaccine was associated with reduced mortality but did not provide sterile immunity.     

Clinical and pathologic responses of American crows (Corvus brachyrhynchos) and fish crows (C ossifragus) to experimental West Nile virus infection

West Nile virus (WNV)-associated disease has a range of clinical manifestations among avian taxa, the reasons for which are not known. Species susceptibility varies within the avian family Corvidae, with estimated mortality rates ranging from 50 to 100%. We examined and compared virologic, immunologic, pathologic, and clinical responses in 2 corvid species, the American crow (Corvus brachyrhynchos) and the fish crow (C ossifragus), following experimental WNV inoculation. Unlike fish crows, which remained clinically normal throughout the study, American crows succumbed to WNV infection subsequent to dehydration, electrolyte and pH imbalances, and delayed or depressed humoral immune responses concurrent with marked, widespread virus replication. Viral titers were approximately 3,000 times greater in blood and 30,000 to 50,000 times greater in other tissues (eg, pancreas and small intestine) in American crows versus fish crows. Histologic lesion patterns and antigen deposition supported the differing clinical outcomes, with greater severity and distribution of lesions and WNV antigen in American crows. Both crow species had multiorgan necrosis and inflammation, although lesions were more frequent, severe, and widespread in American crows, in which the most commonly affected tissues were small intestine, spleen, and liver. American crows also had inflammation of vessels and nerves in multiple tissues, including heart, kidney, and the gastrointestinal tract. WNV antigen was most commonly observed within monocytes, macrophages, and other cells of the reticuloendothelial system of affected tissues. Collectively, the data support that WNV-infected American crows experience uncontrolled systemic infection leading to multiorgan failure and rapid death.     

Serologic evidence of West Nile virus and Usutu virus infections in Eurasian coots in the Netherlands

West Nile virus (WNV) and Usutu virus (USUV) are arboviruses that are maintained in enzootic transmission cycles between mosquitoes and birds and are occasionally transmitted to mammals. As arboviruses are currently expanding their geographic range and emerging in often unpredictable locations, surveillance is considered an important element of preparedness. To determine whether sera collected from resident and migratory birds in the Netherlands as part of avian influenza surveillance would also represent an effective source for proactive arbovirus surveillance, a random selection of such sera was screened for WNV antibodies using a commercial ELISA. In addition, sera of jackdaws and carrion crows captured for previous experimental infection studies were added to the selection. Of the 265 screened serum samples, 27 were found to be WNV–antibody‐positive, and subsequent cross‐neutralization experiments using WNV and USUV confirmed that five serum samples were positive for only WNV‐neutralizing antibodies and seven for only USUV. The positive birds consisted of four Eurasian coots (Fulica atra) and one carrion crow (Corvus corone) for WNV, of which the latter may suggest local presence of the virus, and only Eurasian coots for USUV. As a result, the screening of a small selection of serum samples originally collected for avian influenza surveillance demonstrated a seroprevalence of 1.6% for WNV and 2.8% for USUV, suggesting that this sustained infrastructure could serve as a useful source for future surveillance of arboviruses such as WNV and USUV in the Netherlands.     

Toxoplasma gondii prevalence in Israeli crows and Griffon vultures

A cross-sectional Toxoplasma gondii seroprevalence study was performed on free ranging crows (Corvus cornis, Corvus monedula, Corvus splendens) and Griffon vultures (Gyps fulvus) from Israel in order to assess exposure to this pathogen in scavenger birds that feed on animal carcasses and their possible role in the epidemiology of toxoplasmosis. Using the modified agglutination test (MAT) with a cutoff titer of 1:25, 52 of 122 crows (42.6%) and 40 of 101 Griffon vultures (39.6%) were found to be T. gondii seropositive. Crow T. gondii seroprevalence was significantly higher in northern areas of Israel (p = 0.007) where annual precipitation is higher and annual summer maximum temperatures are lower than in the drier and warmer south. Seroprevalence in crows was positively associated with higher human population densities possibly related to the increased cat population in these areas. PCR analysis of brain extracts from crows resulted in the detection of T. gondii DNA in 1 seropositive crow from northern Israel. Genetic analysis of DNA from the positive crow brain confirmed infection with T. gondii type 2 using a multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP) of the SAG1, 5–3′ SAG2, alt.SAG2, SAG3, BTUB, GRA6, C22-8, c29-2, L358, PK1 and Apico loci. The high T. gondii seroprevalence in these bird species suggests that infected carrion may be responsible for widespread infection of carcass scavenger birds which may further transmit infection to other carnivorous intermediate hosts or feline definitive hosts when consumed post-mortally.     

Pathogenicity of West Nile virus for turkeys

In the fall of 1999, West Nile virus (WNV) was isolated during an outbreak of neurologic disease in humans, horses, and wild and zoological birds in New York, Connecticut, and New Jersey. Turkeys could potentially be a large reservoir for WNV because of the high-density turkey farming and the presence of large wild turkey populations in the eastern seaboard of the United States. Little is known about the pathogenicity of WNV in domestic or wild turkeys. Specific-pathogen-free 3-wk-old turkeys were inoculated subcutaneously with 10(3.3) mean tissue culture infective doses of a WNV strain isolated fromthe index case in a New York crow. No clinical signs were observed in the turkeys over the 21 days of the experiment. One turkey died abruptly at 8 days postinoculation (DPI). Many turkeys developed viremia between 2 and 10 DPI, but the average level of virus was very low, less than needed to efficiently infect mosquitos. Low levels of WNV were detected in feces on 4 and 7 DPI, but no virus was isolated from oropharyngeal swabs. WNV wasnot transmitted from WNV-inoculated to contact-exposed turkeys. All WNV-inoculated poults seroconverted on 7 DPI. In the turkey that died, WNV was not isolated from intestine, myocardium, brain, kidney, or cloacal and oropharyngeal swabs, but sparse viral antigen was demonstrated by immunohistochemistry in the heart and spleen. Turkeys in contact with WNV-inoculated turkeys and sham-inoculated controls lacked WNV specific antibodies,and WNV was not isolated from plasma and cloacal and oropharyngeal swabs. These data suggest that WNV lacks the potential to be a major new disease of turkeys and that turkeys will not be a significant amplifying host for infecting mosquitos.     

Experimental studies of the role of the little raven (Corvus mellori) in surveillance for West Nile virus in Australia

Objective To study the potential role of an Australian corvid, the little raven (Corvus mellori), in the surveillance for exotic West Nile virus (WNV) in Australia. Method In a series of trials, little ravens were infected with WNV (strain 4132 New York 1999) and Kunjin virus (strain K42886) by the intramuscular route. They were observed for 20 days during which blood and swab samples were taken for virus isolation. Tissue samples were taken from ravens humanely killed during the acute infection period, and at the termination of the trials, for virus isolation, histopathology and immunohistochemistry. Results Ravens infected with WNV became mildly ill, but all recovered and seroconverted. Blood virus titres peaked around 3 to 4 days after inoculation at levels between 10^3.0 to 10^7.5 plaque forming units/mL. Virus or viral antigen was detected in spleen, liver, lung, kidney, intestine, testis and ovary by virus isolation and/or immunohistochemistry. WNV was detected in oral and cloacal swabs from 2 to 7 days post inoculation. The molecular and pathogenic characteristics of the inocula were consistent with them being of high virulence, as expected for this isolate. Ravens infected with Kunjin virus developed viraemia and seroconverted, although they did not develop disease. Conclusions Little ravens do not develop severe disease in response to virulent WNV infection and for this reason may not be important sentinel hosts in the event of an outbreak of WNV, as in North America. However, as they have relatively high viraemias, they may be able to support virus cycles.     

Transmission dynamics and changing epidemiology of West Nile virus

West Nile virus (WNV) is a flavivirus that is maintained in a bird-mosquito transmission cycle. Humans, horses and other non-avian vertebrates are usually incidental hosts, but evidence is accumulating that this might not always be the case. Historically, WNV has been associated with asymptomatic infections and sporadic disease outbreaks in humans and horses in Africa, Europe, Asia and Australia. However, since 1994, the virus has caused frequent outbreaks of severe neuroinvasive disease in humans and horses in Europe and the Mediterranean Basin. In 1999, WNV underwent a dramatic expansion of its geographic range, and was reported for the first time in the Western Hemisphere during an outbreak of human and equine encephalitis in New York City. The outbreak was accompanied by extensive and unprecedented avian mortality. Since then, WNV has dispersed across the Western Hemisphere and is now found throughout the USA, Canada, Mexico and the Caribbean, and parts of Central and South America. WNV has been responsible for >27,000 human cases, >25,000 equine cases and hundreds of thousands of avian deaths in the USA but, surprisingly, there have been only sparse reports of WNV disease in vertebrates in the Caribbean and Latin America. This review summarizes our current understanding of WNV with particular emphasis on its transmission dynamics and changing epidemiology.     

Changing patterns of West Nile virus transmission: altered vector competence and host susceptibility

West Nile virus (WNV) is a flavivirus (Flaviviridae) transmitted between Culex spp. mosquitoes and avian hosts. The virus has dramatically expanded its geographic range in the past ten years. Increases in global commerce, climate change, ecological factors and the emergence of novel viral genotypes likely play significant roles in the emergence of this virus; however, the exact mechanism and relative importance of each is uncertain. Previously WNV was primarily associated with febrile illness of children in endemic areas, but it was identified as a cause of neurological disease in humans in 1994. This modulation in disease presentation could be the result of the emergence of a more virulent genotype as well as the progression of the virus into areas in which the age structure of immunologically naïve individuals makes them more susceptible to severe neurological disease. Since its introduction to North America in 1999, a novel WNV genotype has been identified that has been demonstrated to disseminate more rapidly and with greater efficiency at elevated temperatures than the originally introduced strain, indicating the potential importance of temperature as a selective criteria for the emergence of WNV genotypes with increased vectorial capacity. Even prior to the North American introduction, a mutation associated with increased replication in avian hosts, identified to be under adaptive evolutionary pressure, has been identified, indicating that adaptation for increased replication within vertebrate hosts could play a role in increased transmission efficiency. Although stable in its evolutionary structure, WNV has demonstrated the capacity for rapidly adapting to both vertebrate hosts and invertebrate vectors and will likely continue to exploit novel ecological niches as it adapts to novel transmission foci.     

Use of a public telephone hotline to detect urban plague cases

Current methods for vector‐borne disease surveillance are limited by time and cost. To avoid human infections from emerging zoonotic diseases, it is important that the United States develop cost‐effective surveillance systems for these diseases. This study examines the methodology used in the surveillance of a plague epizootic involving tree squirrels (Sciurus niger) in Denver Colorado, during the summer of 2007. A call‐in centre for the public to report dead squirrels was used to direct animal carcass sampling. Staff used these reports to collect squirrel carcasses for the analysis of Yersinia pestis infection. This sampling protocol was analysed at the census tract level using Poisson regression to determine the relationship between higher call volumes in a census tract and the risk of a carcass in that tract testing positive for plague. Over‐sampling owing to call volume‐directed collection was accounted for by including the number of animals collected as the denominator in the model. The risk of finding an additional plague‐positive animal increased as the call volume per census tract increased. The risk in the census tracts with >3 calls a month was significantly higher than that with three or less calls in a month. For tracts with 4–5 calls, the relative risk (RR) of an additional plague‐positive carcass was 10.08 (95% CI 5.46–18.61); for tracts with 6–8 calls, the RR = 5.20 (2.93–9.20); for tracts with 9–11 calls, the RR = 12.80 (5.85–28.03) and tracts with >11 calls had RR = 35.41 (18.60–67.40). Overall, the call‐in centre directed sampling increased the probability of locating plague‐infected carcasses in the known Denver epizootic. Further studies are needed to determine the effectiveness of this methodology at monitoring large‐scale zoonotic disease occurrence in the absence of a recognized epizootic.     

Evaluation of factors associated with positive IgM capture ELISA results in equids with clinical signs compatible with West Nile virus infection: 1,017 cases (2003)

OBJECTIVE: To describe the prevalence of West Nile virus (WNV) infection and evaluate factors associated with positive IgM capture ELISA results in equids with clinical signs compatible with WNV infection. DESIGN: Retrospective case series. SAMPLE POPULATION: Laboratory submission forms from 1,104 equids tested for WNV in Colorado in 2003. PROCEDURES: Submission forms accompanying samples submitted for detection of WNV via IgM capture ELISA were obtained from the Colorado state veterinarian and diagnostic laboratories performing the tests. Data on signalment, clinical signs, history of vaccination against WNV, and assay results were collected from laboratory submission forms. Equids with clinical signs compatible with WNV infection in which IgM capture ELISA results were positive were considered as case equids. RESULTS: 1,104 equids were tested for WNV; 1,017 (92.1%) had clinical signs compatible with WNV infection. Among equids with clinical signs compatible with WNV infection, the odds of testing positive for WNV via IgM capture ELISA were lower in males and in vaccinated equids and higher in equids with moderate and severe illness, compared with females, unvaccinated equids, and equids with mild illness. CONCLUSIONS AND CLINICAL RELEVANCE: Among equids with clinical signs compatible with WNV infection, vaccination against WNV, severity of clinical signs, duration of illness, and region in Colorado were associated with increased risk of having a positive IgM capture ELISA result.     

Epidemic West Nile virus encephalomyelitis: a temperature-dependent, spatial model of disease dynamics

Since first being detected in New York in 1999, West Nile virus (WNV) has spread throughout the United States and more than 20,000 cases of equine WNV encephalomyelitis have been reported. A spatial model of disease occurrence was developed, using data from an outbreak of serologically confirmed disease in an unvaccinated population of horses at 108 locations in northern Indiana between 3 August and 17 October 2002. Daily maximum temperature data were recorded at meteorological stations surrounding the study area. The distribution of the total number of degree-days elapsing between July 4 and the date of diagnosis of each case was best described by a normal distribution (mean = 5243 °F, S.D. = 1047). The days on which the average risk was >25, >50 and >75% were predicted (versus observed) to occur on August 23 (August 9), August 31 (September 2) and September 9 (September 9). The epidemic was predicted to occur 3 days earlier, or 4 days later, than observed if temperatures in the study area were uniformly increased, or decreased, by 5 °F, respectively. Maps indicated that WNV encephalomyelitis risk always remained greater in the northwest quadrant of the study area. Since WNV might exist at a hypoendemic level of infection, and occasionally re-emerge as a cause of epidemics in equine populations, by identifying factors that contributed to this epidemic, the potential impact of future epidemics can be reduced. Such studies rely on a GIS framework, availability of meteorological and possibly remotely sensed data and information on host and landscape factors. An early-warning system for WNV transmission in equine populations could be developed.     

Pathogenesis of West Nile virus lineage 1 and 2 in experimentally infected large falcons

West Nile virus (WNV) is a zoonotic flavivirus that is transmitted by blood-suckling mosquitoes with birds serving as the primary vertebrate reservoir hosts (enzootic cycle). Some bird species like ravens, raptors and jays are highly susceptible and develop deadly encephalitis while others are infected subclinically only. Birds of prey are highly susceptible and show substantial mortality rates following infection. To investigate the WNV pathogenesis in falcons we inoculated twelve large falcons, 6 birds per group, subcutaneously with viruses belonging to two different lineages (lineage 1 strain NY 99 and lineage 2 strain Austria). Three different infection doses were utilized: low (approx. 500 TCID50), intermediate (approx. 4 log10 TCID50) and high (approx. 6 log10 TCID50). Clinical signs were monitored during the course of the experiments lasting 14 and 21 days. All falcons developed viremia for two weeks and shed virus for almost the same period of time. Using quantitative real-time RT-PCR WNV was detected in blood, in cloacal and oropharyngeal swabs and following euthanasia and necropsy of the animals in a variety of neuronal and extraneuronal organs. Antibodies to WNV were first time detected by ELISA and neutralization assay after 6 days post infection (dpi). Pathological findings consistently included splenomegaly, non-suppurative myocarditis, meningoencephalitis and vasculitis. By immunohistochemistry WNV-antigens were demonstrated intralesionally. These results impressively illustrate the devastating and possibly deadly effects of WNV infection in falcons, independent of the genetic lineage and dose of the challenge virus used. Due to the relatively high virus load and long duration of viremia falcons may also be considered competent WNV amplifying hosts, and thus may play a role in the transmission cycle of this zoonotic virus.     

West Nile Virus Transmission in 2008 in North‐Eastern Italy

After 10 years, West Nile virus (WNV) re‐emerged in Italy in August 2008. As on 31 December 2008, the infection affected eight Provinces in three Regions (Emilia Romagna, Veneto, Lombardy), where a total of 794 cases of WNV infection in 251 equine stables were detected on the basis of the clinical signs and as a result of a serological screening in horses living in the area. Only 4.0% (32/794) of the serologically positive animals showed clinical signs, and the 32 clinical cases were reported in 18 different farms. The observed case‐fatality rate was 15.6% (5/32). The confirmed clinical cases were detected from end August to mid October. Significant levels of positivity by RT‐PCR were also observed in magpies (Pica pica) (9.1%, 95% confidence levels: 6.1–13.4%), carrion crows (Corvus corone) (7.4%, 95% confidence levels: 3.6–14.4%) and rock pigeons (Columba livia) (12.9%, 95% confidence levels: 7.6–21.2%).     

Low levels of antibacterial drug resistance expressed by Gram-negative bacteria isolated from poultry carcasses in New Zealand

Abstract

AIM: To provide baseline data on the levels and patterns of antibacterial drug resistance expressed by Gram-negative bacteria isolated from poultry carcasses in New Zealand.

METHODS: Between July and December 2006, isolates of Escherichia coli (n=407) and Salmonella spp. (n=3) originating from carcass-rinse samples were submitted by testing laboratories affiliated to five major poultry processing plants. Isolates of Campylobacter jejuni (n=193) originating from retail poultry carcasses in 2005–2006 were retrieved from the Massey University archives. All isolates underwent disc diffusion susceptibility testing against panels of 12 (Enterobacteriaceae) and six (Campylobacter spp.) antibacterial drugs. Cephalothin-resistance in isolates of E. coli was confirmed using ETest strips, and confirmation of the resistance phenotypes for a subset of C. jejuni isolates used microbroth dilution assays. Patterns within the resistance phenotypes of the isolates were investigated using hierarchical clustering, and logistic regression modelling.

RESULTS: The majority of isolates (71.5% E. coli, 99% C. jejuni, and all three Salmonella spp. isolates) were fully susceptible to the drugs that were tested. Four (1%) E. coli isolates showed resistance to three or more drugs. The proportions of susceptible E. coli differed between the five processing plants. Resistances were detected in E. coli isolates, using disc diffusion to cephalothin (18.2%), ampicillin (4.4%), tetracycline (4.4%) and gentamicin (1.5%). There was an association between cephalothin-resistant isolates of E. coli and decreased susceptibility to gentamicin. Using ETests to ascertain the minimum inhibitory concentrations (MIC) of E. coli for cephalothin gave inconsistent results. One of 193 C. jejuni isolates was resistant to erythromycin, and microbroth dilution assays confirmed that this panel of C. jejuni was generally susceptible to antibacterial drugs.

CONCLUSIONS: The levels of resistance shown by Gram-negative bacteria isolated from chicken carcasses in New Zealand are among the lowest reported around the world. No resistance to extended-spectrum cephalosporin drugs was detected in E. coli, suggesting that CTX-M and AmpC beta-lactamases are rare or absent. Salmonella spp. are rarely isolated from poultry carcasses during routine testing in New Zealand, and the isolates identified during this study were fully susceptible to the drugs tested. A panel of C. jejuni isolates originating from retail poultry carcasses were susceptible to first-line and second-line antibacterial drugs. The use of cephalothin as a marker of resistance to first-generation cephalosporins may not be appropriate for non-type-specific E. coli of animal origin.     

West Nile virus in the United States (1999-2005)

The accidental introduction of West Nile Virus into New York City from the Old World in 1999 resulted in an epidemic in humans, horses, and birds that swept to the west coast in just 3 years. The virus is transmitted by infective mosquitoes among susceptible native birds, which serve as amplifying hosts. Clinical disease occurs in humans and horses, but not enough virus is produced in their blood to infect other mosquitoes; therefore, humans and horses are considered dead-end hosts. Humans can best protect themselves by remaining indoors during periods of high mosquito activity and/or by using recommended repellents. Effective vaccines are available for horses.     

Evaluation of antibody response to vaccination against West Nile virus in thick billed parrots (Rhynchopsitta pachyrhyncha)

West Nile virus (WNV) was first documented in North America in New York City in 1999. Several deaths attributable to WNV have been reported in captive thick-billed parrots (Rhynchopsitta pachyrhyncha), an endangered psittacine native to North America. The serologic responses in 12 captive adult thick-billed parrots after a series of three initial WNV vaccine injections with annual boosters over 6 yr was evaluated. In addition, the serologic responses of 11 thick-billed parrot chicks following an initial vaccination series to determine if there were seroconversions were also reported. Most adults (67%) had seroconverted after 5 yr of annual vaccination, with a median titer of 1:80 (range 1:40-1:160) for those that seroconverted. After the first year, birds were likely naturally exposed to WNV, which limited interpretation of titers. None of the chicks seroconverted during the initial three-vaccine series; only two of four chicks (50%) had seroconverted when tested at the 1-yr yearly booster, and at 2 yr, three of four chicks had seroconverted. Although some birds had detectable antibody titers, it is unclear whether this vaccine can reliably provide protection against WNV in thick-billed parrots.     

Simulation model for Campylobacter cross-contamination during poultry processing at slaughterhouses

Broiler meat is regarded as the most common source of Campylobacter infection and risk management measures are required to reduce broiler meat contamination. Among the quantitative risk assessments for Campylobacter in broiler meat, evaluation of the poultry processing stage is particularly important for predicting the contamination level of broiler meat and the effects of control measures. In this study, we built a simulation model for cross‐contamination during poultry processing focusing on Campylobacter contamination at the individual carcass level. Using this model, we examined changes in the prevalence of contaminated carcasses and the number of Campylobacter per carcass after processing. As a result, it was found that the prevalence and number of Campylobacter after processing were largely influenced by the number of Campylobacter on the contaminated carcasses before processing. In the baseline model, where it was assumed that the mean number of Campylobacter on contaminated carcasses before processing was 4.7 log₁₀ cfu per carcass, the prevalence after processing was less than that before processing. Although the median value of Campylobacter on contaminated carcasses was reduced after processing, the distributions after processing became wider and the upper limit of the 95% credible interval of Campylobacter on contaminated carcasses remained elevated. The individual‐based simulation model can trace individual level changes considering discrete interactions, while models tracing mean values cannot handle these interactions in detail. The individual‐based approach is considered useful for modelling cross‐contamination among individual carcasses during poultry processing.