Epidemiology and economic impact of lumpy skin disease of cattle in Mymensingh and Gaibandha districts of Bangladesh.

Lumpy skin disease (LSD) is an emerging viral disease of cattle that negatively impacts livestock by reducing animals' production and increasing management costs. Like other countries, Bangladesh has also faced the ominous effects of LSD. Because of this pressing concern, the present study was undertaken to determine the attack rate and risk factors along with economic losses of the recent LSD outbreaks from October 2019 to June 2020 in Bangladesh's Mymensingh and Gaibandha districts. A total of 1187 cattle from 150 randomly selected cattle farms were surveyed. The diagnosis was made both clinically and molecularly. The overall attack rate, mortality and case fatality were 34%, 1.10% and 3.23%, respectively, but in calves up to 1 month of age, the attack rate, mortality and case-fatality were 41.6%, 12.5% and 30%, respectively. In risk factor analysis using multiple logistic regression, the following factors were significantly (p < .05) associated with LSD occurrence: no administration of goat pox vaccine, frequency of goat pox vaccination - single dose, introduction of new cattle, land type (low land and medium-high land), use of common syringe and needle, and use of common utensils. The average economic loss per case was 9384.41 BDT (110.40 US $). The higher economic loss per case was accounted in Mymensingh district (10248.91 BDT ≈ 120.58 US $) than Gaibandha district (8211.52 BDT ≈ 96.61 US $). The loss was higher in crossbred cattle (9709.58 BDT ≈ 114.23 US $) compared to indigenous cattle (7595.94 BDT ≈ 89.36 US $). The total estimated annual loss due to LSD in Mymensingh and Gaibandha districts was 7763.25 million BDT (91.33 million US $). In Gaibandha district, the estimated annual loss was 2666.20 million BDT (31.37 million US $) while in Mymensingh district it was 5097.05 million BDT (59.97 million US $). Findings of the study could provide useful epidemiological data with losses incurred due to LSD to the livestock owners, field veterinarians and government-level livestock regulators, which will help to formulate prevention and control strategies and minimize the negative impact of LSD's probable return or spread.

Nitric oxide mediates antimicrobial peptide gene expression by activating eicosanoid signaling.

Nitric oxide (NO) mediates both cellular and humoral immune responses in insects. Its mediation of cellular immune responses uses eicosanoids as a downstream signal. However, the cross-talk with two immune mediators was not known in humoral immune responses. This study focuses on cross-talk between two immune mediators in inducing gene expression of anti-microbial peptides (AMPs) of a lepidopteran insect, Spodoptera exigua. Up-regulation of eight AMPs was observed in S. exigua against bacterial challenge. However, the AMP induction was suppressed by injection of an NO synthase inhibitor, L-NAME, while little expressional change was observed on injecting its enantiomer, D-NAME. The functional association between NO biosynthesis and AMP gene expression was further supported by RNA interference (RNAi) against NO synthase (SeNOS), which suppressed AMP gene expression under the immune challenge. The AMP induction was also mimicked by NO alone because injecting an NO analog, SNAP, without bacterial challenge significantly induced the AMP gene expression. Interestingly, an eicosanoid biosynthesis inhibitor, dexamethasone (DEX), suppressed the NO induction of AMP expression. The inhibitory activity of DEX was reversed by the addition of arachidonic acid, a precursor of eicosanoid biosynthesis. AMP expression of S. exigua was also controlled by the Toll/IMD signal pathway. The RNAi of Toll receptors or Relish suppressed AMP gene expression by suppressing NO levels and subsequently reducing PLA2 enzyme activity. These results suggest that eicosanoids are a downstream signal of NO mediation of AMP expression against bacterial challenge.

Nitric Oxide Mediates Insect Cellular Immunity via Phospholipase A2 Activation.

After infection or invasion is recognized, biochemical mediators act in signaling insect immune functions. These include biogenic amines, insect cytokines, eicosanoids, and nitric oxide (NO). Treating insects or isolated hemocyte populations with different mediators often leads to similar results. Separate treatments with an insect cytokine, 2 biogenic amines, and an eicosanoid lead to a single result, hemocyte spreading, understood in terms of intracellular cross-talk among these signaling systems. This study focuses on the cross-talk between NO and eicosanoid signaling in our model insect, Spodoptera exigua. Bacterial injection increased NO concentrations in the larval hemocytes and fat body, and RNA interference (RNAi) of the S. exigua NO synthase (NOS) gene suppressed NO concentrations. RNAi treatment also led to a significant reduction in hemocyte nodulation following bacterial injection. Similar RNAi treatments led to significantly reduced PLA2 activities in the hemocytes and fat body compared to control larvae. Injection of L-NAME also prevented the induction of PLA2 activity following bacterial challenge. An injected NO donor, S-nitroso-N-acetyl-DL-penicillamine, increased PLA2 activity in a dose-dependent manner. However, eicosanoids did not influence NO concentrations in immune-challenged larvae. We infer that NO and eicosanoid signaling operate via cross-talk mechanisms in which the elevated NO concentrations activate PLA2 and eicosanoid biosynthesis, which finally mediates various immune responses.

A novel calcium-independent phospholipase A2 and its physiological roles in development and immunity of a lepidopteran insect, Spodoptera exigua.

Phospholipase A2 (PLA2) catalyzes hydrolysis of ester linkage at sn-2 position of phospholipids. At least 15 groups (I-XV) of PLA2 gene superfamily are associated with various physiological processes such as digestion, secretion, immunity, and maintenance of membrane integrity. This study suggests that various insects encode putative Group VI PLA2s representing intracellular and calcium-independent PLA2s (iPLA2). These insect iPLA2s are separated into at least two subgroups: iPLA2A (Group VIA-like) and iPLA2B (non-Group VIA). Most insects encode genes of iPLA2B type, although their biological functions are currently unknown. This study predicted a novel iPLA2 from Spodoptera exigua (a lepidopteran insect) (SeiPLA2B) and analyzed its physiological functions by RNA interference (RNAi). SeiPLA2B encodes 336 amino acid sequence with a predicted size of about 36.6 kDa and an isoelectric point at pH 8.61. It possesses a lipase catalytic site, but does not have ankyrin repeats in the amino terminal region. Phylogenetic analysis indicated that SeiPLA2B was clustered with other Group VI iPLA2s, in which SeiPLA2B was closely associated with Group VIF gene while SeiPLA2A was closely related to Group VIA gene. SeiPLA2B was expressed in all developmental stages of S. exigua. In larval stage, SeiPLA2B was expressed in fat body, hemocyte, and epidermis, but not in digestive tract. SeiPLA2B RNAi significantly reduced PLA2 enzyme activities and resulted in developmental retardation and immunosuppression. Though RNAi treatment did not significantly change fatty acid composition in fat body lipids, it significantly increased lipid peroxidation. Taken together, our results suggest that SeiPLA2B plays important roles in the development and immunity of S. exigua.

Specific inhibition of Xenorhabdus hominickii, an entomopathogenic bacterium, against different types of host insect phospholipase A2.

Phospholipase A2 (PLA2) hydrolyzes ester bond of phospholipids at the sn-2 position to release free fatty acid and lysophospholipids. Some PLA2s preferentially release arachidonic acid which is subsequently oxygenated into eicosanoids to mediate immune responses in insects. Xenorhabdus hominickii is an entomopathogenic bacterium that can suppress insect immunity by inhibiting PLA2 activity. However, little is known about target PLA2 types inhibited by X. hominickii. Therefore, the objective of this study was to determine PLA2 types in the host insect, Spodoptera exigua using specific inhibitors. All developmental stages of S. exigua possessed significant PLA2 activities, with late larval stages showing relatively higher PLA2 activities. In different larval tissues, hemocytes had higher PLA2 activities than fat body, gut, or epidermis. Various developmental and tissue extracts exhibited differential susceptibilities to three different PLA2 inhibitors. Late larva-to-adult stages were highly susceptible to all three different types of PLA2 inhibitors. In contrast, extracts from egg and young larval stages were not susceptible to secretory PLA2 (sPLA2) or calcium-independent cellular PLA2 (iPLA2) inhibitors, although they were susceptible to a calcium-dependent cellular PLA2 (cPLA2) inhibitor in a dose-dependent manner. Different tissues of fifth instars exhibited variation in susceptibility to inhibitors, with epidermal tissue being sensitive to cPLA2 inhibitor only while other tissues were sensitive to all three types of inhibitors. Bacterial challenge with heat-killed X. hominickii significantly increased PLA2 activity. However, live bacteria suppressed the induction of PLA2 activity. An organic extract of X. hominickii-culture broth inhibited the susceptibility of S. exigua to sPLA2- and iPLA2- specific inhibitors, but not to cPLA2-specific inhibitor. Oxindole, a component of the organic extract, exhibited an inhibitory pattern similar to the organic extract. Taken together, our results indicate that S. exigua possesses different PLA2 types and that X. hominickii can inhibit PLA2s susceptible to sPLA2- and iPLA2- specific inhibitors.

An entomopathogenic bacterium, Xenorhabdus hominickii ANU101, produces oxindole and suppresses host insect immune response by inhibiting eicosanoid biosynthesis.

An entomopathogenic bacterium, Xenorhabdus hominickii ANU101, was isolated from an entomopathogenic nematode, Steinernema monticolum. X. hominickii exhibited significant insecticidal activities at ≥6.6×102 colony-forming units per larva against a lepidopteran insect, Spodoptera exigua with hemocoelic injection. The insecticidal activity of X. hominickii was reduced by an addition of arachidonic acid (AA, a catalytic product of PLA2), but enhanced by an addition by dexamethasone (DEX, a specific inhibitor of PLA2). S. exigua could defend the bacterial infection by forming hemocyte nodules. However, live X. hominickii significantly reduced the hemocytic nodulation compared to similar treatment with heat-killed X. hominickii. An addition of AA to live X. hominickii significantly rescued the immunosuppression. X. hominickii also inhibited phenoloxidase activity in hemolymph of S. exigua larvae. Furthermore, the bacteria suppressed gene expressions of antimicrobial peptides, such as attacin-1, attacin-2, defensin, gallerimycin and transferrin-1 of S. exigua. An organic extract of X. hominickii-cultured broth with ethyl acetate possessed oxindole and significantly suppressed hemocyte nodulation. Again, an addition of AA diminished the inhibitory activity of the organic extract against hemocyte nodulation. Oxindole alone inhibited hemocyte nodulation and PLA2 enzyme activity. These results suggest that the entomopathogenicity of X. hominickii comes from its inhibitory activity against eicosanoid biosynthesis of target insects.

Identification and bacterial characteristics of Xenorhabdus hominickii ANU101 from an entomopathogenic nematode, Steinernema monticolum.

An entomopathogenic nematode, Steinernema monticolum, was collected in Korea. Its identity was confirmed by morphological and molecular characters. Its symbiotic bacterium, Xenorhabdus hominickii ANU101, was isolated and assessed in terms of bacterial characteristics. Sixty-eight different carbon sources were utilized by X. hominickii ANU101 out of 95 different sources from a Biolog assay. Compared to other Xenorhabdus species, X. hominickii ANU101 was relatively susceptible to high temperatures and did not grow above 34°C. Furthermore, its growth rate was much slower than other Xenorhabdus species. X. hominickii exhibited insecticidal activities against coleopteran, dipteran, and lepidopteran insect pests. The bacterial virulence was not correlated with its host nematode virulence with respect to relative insecticidal activity against target insects. X. hominickii ANU101 exhibited antibiotics tolerance. The bacterium possesses four different plasmids (Xh-P1 (104,132bp), Xh-P2 (95,975bp), Xh-P3 (88,536bp), and Xh-P4 (11,403bp)) and encodes 332 open reading frames. Subsequent predicted genes include toxin/antitoxins comprising a multidrug export ATP-binding/permease. This study reports bacterial characters of X. hominickii and its entomopathogenicity.