The Acquisition and Retention of Lumpy Skin Disease Virus by Blood-Feeding Insects Is Influenced by the Source of Virus, the Insect Body Part, and the Time since Feeding

Abstract

Lumpy skin disease virus (LSDV) is a poxvirus that causes severe systemic disease in cattle and is spread by mechanical arthropod-borne transmission. This study quantified the acquisition and retention of LSDV by four species of Diptera (Stomoxys calcitrans, Aedes aegypti, Culex quinquefasciatus, and Culicoides nubeculosus) from cutaneous lesions, normal skin, and blood from a clinically affected animal. The acquisition and retention of LSDV by Ae. aegypti from an artificial membrane feeding system was also examined. Mathematical models of the data were generated to identify the parameters which influence insect acquisition and retention of LSDV. For all four insect species, the probability of acquiring LSDV was substantially greater when feeding on a lesion compared with feeding on normal skin or blood from a clinically affected animal. After feeding on a skin lesion LSDV was retained on the proboscis for a similar length of time (around 9 days) for all four species and for a shorter time in the rest of the body, ranging from 2.2 to 6.4 days. Acquisition and retention of LSDV by Ae. aegypti after feeding on an artificial membrane feeding system that contained a high titer of LSDV was comparable to feeding on a skin lesion on a clinically affected animal, supporting the use of this laboratory model as a replacement for some animal studies. This work reveals that the cutaneous lesions of LSD provide the high-titer source required for acquisition of the virus by insects, thereby enabling the mechanical vector-borne transmission. IMPORTANCE Lumpy skin disease virus (LSDV) is a high consequence pathogen of cattle that is rapidly expanding its geographical boundaries into new regions such as Europe and Asia. This expansion is promoted by the mechanical transmission of the virus via hematogenous arthropods. This study quantifies the acquisition and retention of LSDV by four species of blood-feeding insects and reveals that the cutaneous lesions of LSD provide the high titer virus source necessary for virus acquisition by the insects. An artificial membrane feeding system containing a high titer of LSDV was shown to be comparable to a skin lesion on a clinically affected animal when used as a virus source. This promotes the use of these laboratory-based systems as replacements for some animal studies. Overall, this work advances our understanding of the mechanical vector-borne transmission of LSDV and provides evidence to support the design of more effective disease control programmes.

Keywords: Aedes aegypti; Culex quinquefasciatus; Culicoides nubeculosus; Stomoxys calcitrans; control; flies; lumpy skin disease; midges; mosquitoes; poxvirus; transmission; vector.

FIG 1

Clinical signs and levels of lumpy skin disease (LSD) viral DNA and virus in the donor calf. (A) Rectal temperature (°C). (B) Gross pathology of experimental LSD in the donor calf. (C) Levels of viral DNA in blood (log₁₀ copies/mL). (D) Levels of viral DNA (log₁₀ copies/1 mm skin) in lesions (magenta) and normal skin (red) to which different insects were exposed: Aedes aegypti (circles); Culex quinquefasciatus (up-triangles); Culicoides nubeculosus (down-triangles); and Stomoxys calcitrans (diamonds). (E) Example of a lesion on the calf and areas where samples were taken (red circles). (F) Levels of infectious virus (log₁₀ PFU/g) in samples taken from the center of a lesion (R), adjacent to a lesion (N), or approximately 10 mm from the lesion (P).

FIG 2

The proportion of insect parts positive for lumpy skin disease viral DNA depends on virus source, body part tested, and time post-feeding for four species of biting insect. Each plot shows the observed proportion of positive insects (symbols) and the posterior median for the expected proportion of positive insects (lines). Virus source is indicated by color and symbol: normal skin of a clinical calf (red up-triangles), a lesion on a clinical calf (magenta circles), or blood from a clinical calf via an artificial membrane feeding system (blue down-triangles). The body part tested (proboscis, head/thorax, abdomen, or body) is indicated in the y axis label.

FIG 3

Levels of retained lumpy skin viral DNA depends on virus source, body part tested, and time post-feeding for four species of biting insect. Each plot shows the level of viral DNA retained in each part (log₁₀ copy number/part; symbols) and the mean level retained (horizontal lines). Virus source is indicated by color and symbol: normal skin of a clinical calf (red up-triangles), a lesion on a clinical calf (magenta circles), or blood from a clinical calf via an artifical membrane feeding system (blue down-triangles). The body part tested (proboscis [prob.], head/thorax [head], abdomen [abd.], or body) is indicated in the y axis label.

FIG 4

Relationship between level of lumpy skin disease viral DNA and the probability of virus acquisition. Each plot shows the dose-response relationship between the probability of an insect part (proboscis, head/thorax, abdomen, or body) being positive and the level of viral DNA to which the insect was exposed at feeding (log₁₀ copy number/mL). Four species of insect (indicated at the top of each column) were tested at 0, 2, 4, and 8 days post-feeding (dpf, rows). Plots show the observed proportion of positive proboscises (orange circles), head/thoraxes (purple up-triangles), abdomens (green down-triangles), and bodies (blue diamonds) and the posterior median probability of an insect part being positive (lines: proboscis, orange; head/thorax, purple; abdomen, green; body, blue).

FIG 5

Acquisition and retention of lumpy skin viral DNA in different body parts of Aedes aegypti after feeding ex vivo: proboscis (top row), head/thorax (middle row), or abdomen (bottom row). The first column shows the proportion of insect parts positive for viral DNA. Each plot shows the observed proportion of positive parts (symbols) and the posterior median for the expected proportion of positive parts (lines). The second, third, and fourth columns show the levels of lumpy skin viral DNA (log₁₀ copy number/part) retained in each part. The circles in each plot show the levels for individual parts and the solid black line indicates the mean. In each panel, the source of virus is indicated by color: lesion ex vivo (blue); blood/virus mixture, high titer (orange); or blood/virus mixture, low titer (purple).