Mosquito-Independent Transmission of West Nile virus in Farmed Saltwater Crocodiles (Crocodylus porosus)

Abstract

West Nile virus, Kunjin strain (WNV KUN ) is endemic in Northern Australia, but rarely causes clinical disease in humans and horses. Recently, WNV KUN genomic material was detected in cutaneous lesions of farmed saltwater crocodiles (Crocodylus porosus), but live virus could not be isolated, begging the question of the pathogenesis of these lesions. Crocodile hatchlings were experimentally infected with either 10 5 (n = 10) or 10 4 (n = 11) TCID 50 -doses of WNV KUN and each group co-housed with six uninfected hatchlings in a mosquito-free facility. Seven hatchlings were mock-infected and housed separately. Each crocodile was rotationally examined and blood-sampled every third day over a 3-week period. Eleven animals, including three crocodiles developing typical skin lesions, were culled and sampled 21 days post-infection (dpi). The remaining hatchlings were blood-sampled fortnightly until experimental endpoint 87 dpi. All hatchlings remained free of overt clinical disease, apart from skin lesions, throughout the experiment. Viremia was detected by qRT-PCR in infected animals during 2-17 dpi and in-contact animals 11-21 dpi, indicating horizontal mosquito-independent transmission. Detection of viral genome in tank-water as well as oral and cloacal swabs, collected on multiple days, suggests that shedding into pen-water and subsequent mucosal infection is the most likely route. All inoculated animals and some in-contact animals developed virus-neutralizing antibodies detectable from 17 dpi. Virus-neutralizing antibody titers continued to increase in exposed animals until the experimental endpoint, suggestive of persisting viral antigen. However, no viral antigen was detected by immunohistochemistry in any tissue sample, including from skin and intestine. While this study confirmed that infection of saltwater crocodiles with WNV KUN was associated with the formation of skin lesions, we were unable to elucidate the pathogenesis of these lesions or the nidus of viral persistence. Our results nevertheless suggest that prevention of WNV KUN infection and induction of skin lesions in farmed crocodiles

West Nile Virus: An Update on Pathobiology, Epidemiology, Diagnostics, Control and “One Health” Implications

“…The latter are mainly infected during a mosquito blood meal, if the mosquito saliva titre exceeds 10 4 TCID 50 /mL and may then develop clinical disease [ 42 ] ( Figure 2 ). Final hosts are generally dead-end-hosts, except for the crocodilians which, unlike other final hosts, also amplify the virus [ 39 , 41 ]. Competent mosquito vectors acquire the virus from a viraemic vertebrate host during their blood meal.…”

“…These modes of transmission were first reported during the first WNV outbreak in the USA [ 173 , 174 , 175 , 176 , 177 , 178 ]. Oral-faecal route of transmission has also been confirmed in American alligators and saltwater crocodiles [ 39 , 40 , 41 , 179 ]. Contact transmission in commercial geese farming has also been documented.…”

“…Although bird species are generally WNV reservoirs, investigations conducted during the WNV outbreak between 1999 and 2001 in the USA revealed Corvus species are the most susceptible to the diseases and the main amplifier [ 226 , 227 , 228 , 229 ]. Following the 1999 WNV outbreak in American alligators in the Americas and WNV associated “pix” lesions in saltwater crocodiles in Australia, experimental studies suggested that American alligators and saltwater crocodiles are also WNV amplifiers with high enough titres in their blood to potentially transmit the virus to mosquitoes [ 39 , 41 ].…”

“…However, these lesions are neither specific nor pathognomonic to WNV. Interestingly, although WNV genomic RNA was detected in both oral and cloacal swabs of saltwater crocodiles experimentally infected with WNV KUN as well as in their un-infected pen-mates, C. porosus does not present with gastrointestinal clinical signs or pathological changes, and it remains unknown where exactly in the alimentary tract the virus is replicating in these animals [ 41 ].…”

“…WNV is transmitted by a mosquito vector of the genus Culex through hematophagy [ 30 , 31 , 32 ]. However, some other ways of transmission, including ingestion, aerosol, and direct contact, have been reported in experimental settings [ 33 , 34 ], in humans (intrauterine and breastfeeding transmission) [ 35 , 36 , 37 ], and recently also in farmed alligators and crocodiles [ 38 , 39 , 40 , 41 ]. To date, only one case of vertical transmission has been reported in humans [ 36 , 42 , 43 ].…”