Is Galleria mellonella model a good alternative to study virulence in Staphylococcus aureus from bovine mastitis?

Staphylococcus aureus is one of the agents of bovine mastitis of hardest control due to a complex pathogenesis comprising a variety of virulence factors, which ensures its persistence in the mammary gland, causing significant health and economic losses. Therefore, understanding the pathogenesis of this agent is imperative. Galleria mellonella has stood out as an invertebrate animal model for the study of infectious diseases that affect several hosts. This work aimed to evaluate G. mellonella larvae as an experimental model for the study of virulence phenotypes in an S. aureus population isolated from bovine mastitis. Thirty genetically divergent S. aureus strains were chosen based on PFGE analysis. After experimental infection, larvae survival rates, bacterial growth in hemolymph, melanization intensity of the dorsal vessel, and histological characteristics of the infected tissues were evaluated. The G. mellonella model showed a clear diversity in the S. aureus pathogenicity pattern, allowing the differentiation of strains with virulence phenotypes ranging from high to low degrees. Histological analysis confirmed that the strains tested were capable of inducing the formation of nodules and melanization spots in the dorsal vessels of the larvae in different magnitudes. The strains 16S-717, 19C-828, and 31S-1443 presented the highest virulence intensity among the bacteria tested and will be used further for the generation of S. aureus mutant populations to prospect genetic targets aimed to develop control strategies of bovine mastitis. Altogether, our results suggest that G. mellonella is an attractive and low-cost animal model for characterizing virulence phenotypes of large S. aureus populations.

From the laboratory to the field: efficacy of entomopathogenic nematodes to control the cattle tick.

BACKGROUND: The control of ticks is challenged by the resistance of tick populations to chemical acaricides. In this study, we evaluated, under laboratory conditions, the efficacy of Heterorhabditis bacteriophora against Rhipicephalus (Boophilus) microplus engorged females with varying body weights (150, 200, 250, 300 or 350 mg per female) or from eight different geographical populations. We also determined the efficacy of H. bacteriophora for tick control under field conditions. RESULTS: R. microplus engorged females with varying body weights exposed to 150 juveniles of H. bacteriophora resulted in a high control efficacy (97.5% to 98.4%). Tests with females from different geographical populations comprised eight tick strains treated with H. bacteriophora and their respective control groups. The biological parameters of females exposed to nematode treatments did not differ significantly and resulted in 89% to 99% of control efficacy. Trials conducted under field conditions were performed in field plots with Megathyrsus maximus grass. Treatment groups received eight cadavers of Tenebrio molitor fully colonized with H. bacteriophora at 1 week prior to the release of female ticks, whereas control groups were untreated. On the first day of the experiment, six engorged females were distributed in each plot. On day 42 and day 63, the apical portion of the grasses with R. microplus larvae were collected and quantified. The population of R. microplus larvae was reduced up to 73.1% in plots treated with H. bacteriophora at day 63 after treatment. CONCLUSION: R. microplus engorged females with varying body weights or from different geographical populations were highly susceptible to H. bacteriophora. The field test demonstrated the efficacy of H. bacteriophora in reducing R. microplus larvae in infested pastures. © 2022 Society of Chemical Industry.

Combination of synthetic acaricides with (E)-cinnamaldehyde to control Rhipicephalus microplus.

This work had the objectives to (1) evaluate the susceptibility of various Rhipicephalus microplus populations to commercial acaricides, and (2) select commercial acaricides (50-80% effective) and evaluate the effects of binary combinations of the phenylpropanoid (E)-cinnamaldehyde with selected commercial acaricides to control R. microplus under laboratory and field conditions. Using adult immersion tests with 116 populations and 14 commercial acaricides, products showing 50-80% effectiveness (percent control) with the lowest number of active ingredients were selected. Acaricides containing amitraz or chlorfenvinphos were tested in combination with (E)-cinnamaldehyde on a field population (strain CM). We found that (E)-cinnamaldehyde enhanced the activity of both commercial acaricides against R. microplus larvae; however, the enhancement was more accentuated when using amitraz. Experiments combining (E)-cinnamaldehyde + amitraz on unfed larvae and engorged females from another population (strain Gyn) were performed, verifying (E)-cinnamaldehyde enhanced the activity of amitraz. In the field experiment, the application of (E)-cinnamaldehyde appeared toxic to the tick hosts (cattle). We concluded that (E)-cinnamaldehyde enhanced the activity of amitraz against unfed larvae and engorged females of R. microplus; however, in the field test this phenylpropanoid caused intoxication in the cattle. Studies searching for new combinations of compounds from essential oils with amitraz deserve attention, as well as studies to develop formulations using amitraz + (E)-cinnamaldehyde that will be efficient and will not have toxic effects in cattle.

Acaricidal activity of carvacrol and thymol on acaricide-resistant Rhipicephalus microplus (Acari: Ixodidae) populations and combination with cypermethrin: Is there cross-resistance and synergism?

This study evaluated the acaricidal activity of thymol and carvacrol on Rhipicephalus microplus populations with different resistance profiles and investigated the synergistic effect of combinations of these monoterpenes with cypermethrin. The adult immersion test (AIT) was used to characterize the susceptibility of tick populations (45 field populations) to synthetic acaricides: deltamethrin, amitraz and chlorfenvinphos. The larval packet test (LPT) was used to determine the LC50 values for thymol (25 tick populations) and carvacrol (20 tick populations). The susceptible strain Porto Alegre (POA) was used as a reference for calculating the resistance ratio (RR). Subsequently, larval immersion tests (LIT) were performed with combinations of cypermethrin with thymol or carvacrol to assess a synergistic effect. In the AIT, deltamethrin showed efficacy > 90% in one (2.2%) population tested (mean: 12.1 and 11.1 for populations 1-25 and 26-40, respectively), whereas amitraz and chlorfenvinphos showed efficacy > 90% for two (4.4%) populations (mean: 61.3 and 47.3 for populations 1-25 and 26-40, respectively) and eight (17.7%) populations (mean: 69.7 and 59.7 for populations 1-25 and 26-40, respectively). In the LPT, the LC50 values for thymol and carvacrol varied from 0.67 to 2.12 mg/mL and 0.55-3.21 mg/mL, with an average LC50 for populations of 1.49 and 1.75 mg/mL, respectively. For thymol, no resistance was observed in any of the populations, values of RR50 > 1.5. There was no correlation between the LC50 values for thymol and the efficacy of the chemical acaricides tested. Regarding carvacrol, for only one tick population had the value of RR50 > 1.5, indicating an incipient resistance. No correlation was observed between the LC50 values for carvacrol and the efficacy of tested acaricides. The combination of thymol and carvacrol with cypermethrin showed a synergistic effect in the resistant population (Jaguar - thymol 4.19 and carvacrol 3.67), and no synergistic interaction were showed in the susceptible population. Answering the questions we conclude that: 1 - The comparison between the LC50 values for thymol and carvacrol in field populations and the susceptible strain POA suggests the absence of cross-resistance (ticks and terpenes), and the differences between the LC50 values for thymol and carvacrol in the different R. microplus populations are inherent to the characteristics of each population tested; 2 - the combination of thymol or carvacrol with cypermethrin showed a synergistic effect with different activity according to the population of ticks.

Acaricidal activity of (E)-cinnamaldehyde and α-bisabolol on populations of Rhipicephalus microplus (Acari: Ixodidae) with different resistance profiles.

This study aimed to investigate the acaricidal activity of (E)-cinnamaldehyde and α-bisabolol on populations of Rhipicephalus microplus with different resistance profiles. The adult immersion test (AIT) was used to characterize the susceptibility of tick populations (50 field populations) to synthetic acaricides: deltamethrin, amitraz, and chlorfenvinphos. The larval packet test (LPT) was used to determine the LC50 values for (E)-cinnamaldehyde (populations 1-25) and α-bisabolol (populations 26-50) at the concentrations of 0.31, 0.62, 1.25, 2.0, 2.5, 5.0 and 10.0 mg/mL. The susceptible strain Porto Alegre (POA) was used as a reference for calculating the resistance ratio (RR). In the AIT, deltamethrin did not show efficacy >95 % for any of the populations, whereas amitraz and chlorfenvinphos have presented efficacy >95 % for three (6 %) and 15 (30 %) populations, respectively. In the LPT, the LC50 values of (E)-cinnamaldehyde and α-bisabolol varied from 0.23 to 2.36 mg/mL and 1.57-3.01 mg/mL, respectively. The RR50 for (E)-cinnamaldehyde showed 20 (80 %) populations with values <1.0 and no population with values>1.5. As for α-bisabolol, only two (8%) populations have presented RR50 <1.0, whereas three (12 %) populations showed incipient resistance to this sesquiterpene (RR50 between 1.5 and 2.0). The results indicate that all studied tick populations showed low susceptibility to at least one of the commercial acaricides tested. In addition, comparison between the LC50 values of (E)-cinnamaldehyde and α-bisabolol for the field populations and the susceptible strain POA suggests that there is no cross-resistance of (E)-cinnamaldehyde and α-bisabolol for the tick populations evaluated, and that the differences in the LC50 values are due to population variations.

Entomopathogenic nematodes in pharmaceutical formulations for Rhipicephalus microplus (Acari: Ixodidae) control: In vitro evaluation of compatibility, thermotolerance, and efficiency.

This work aimed to investigate candidate biological control agents against the cattle tick Rhipicephalus microplus by studying three entomopathogenic nematodes (EPN) isolates from two species, Heterorhabditis bacteriophora (isolates LPP30 and HP88) and Heterorhabditis baujardi (isolate LPP7). The study comprised the pharmacotechnical development of four test formulations, and their biological evaluation, in three stages: 1) selection of the fittest EPN isolate at 16 ± 1 °C after 120 h of incorporation in the formulations; 2) determination of the effects of temperature and incubation time on the survival of the selected isolate in formulations; 3) evaluation of the in vitro efficacy (infectivity) of the selected EPN formulations and their components against R. microplus females after 72 h exposure. H. baujardi LPP7 presented the highest survival rates in 0.1% carboxymethyl cellulose sodium (CMC) formulations (100% at 25 °C, up to 10 days after incorporation). At the highest tested temperature (34.2 °C), 54% of LPP7 survived for up to 72 h in CMC formulations. The infectivity of LPP7-containing formulation against R. microplus was 99%, reducing oviposition, egg production index and larval hatching. Considering the pressing need for safer acaricide products, incorporation of EPNs in pharmaceutical formulations is presented as a promising approach for the biological control of cattle tick infestations, potentially reducing environmental impact and resistance development associated with chemical acaricides.

Microarray analysis of tick-infested skin in resistant and susceptible cattle confirms the role of inflammatory pathways in immune activation and larval rejection.

Tick bites promote activation of an inflammatory process that is influenced by bovine genetic composition and its history of previous exposure. Taurine and indicine breeds are known to differ on its immune response development against Rhipicephalus microplus. Nevertheless, further investigation about the complex molecular pathways involved in the development of immune response to tick infestation in cattle presenting the same genetic background is mandatory. The aim of this work was to access the early immune response triggered by R. microplus larvae attachment in previously selected resistant and susceptible animals in a bovine F2 population derived from Gyr (Bos indicus)×Holstein (Bos taurus) crosses. Microarray data analysis of RNA samples from tick infested skin was used to evaluate the gene expression at 0, 24 and 48h after R. microplus larvae attachment. Our experimental design allowed us to deeply explore the immune response related to R. microplus infestation avoiding the innate differences between these breeds. The differentially expressed genes found reveal networks and pathways that suggest a key role of lipid metabolism in inflammation control and impairment of tick infestation in resistant animals. Acute phase response also seems to be impaired in susceptible animals. These results provide new insights about early immune response against ticks and raise the possibility of using immunomodulation processes to improve and develop novel tools for tick control.

Comparative efficiency of two models of CO2 traps in the collection of free-living stages of ixodides.

Traps using carbon dioxide (CO(2)) as a chemical attractant are known to be effective when capturing nymphs and adults of some free-living tick species such as Amblyomma cajennense and Amblyomma parvum. Despite the fact that the main source of CO(2) is dry ice, the chemical trap which uses 20 % lactic acid (C(3)H(6)O(3)) and calcium carbonate (CaCO(3)) has been tested as an alternative source of CO(2) whenever it is difficult to obtain dry ice. The objective of this paper was to test and compare the efficiency of these two models of traps during the study of population dynamics of A. cajennense and Amblyomma dubitatum in Coronel Pacheco, Minas Gerais, Brazil. Within the period comprising May 2006 to April 2008, eight CO(2) traps, of which four were dry ice and four chemical, were put in the pasture every 14 days at preestablished areas over a 1.0-m(2) white cotton flannel cloth with a capture dispositive which constituted of double-sided adhesive tapes fixed onto the four corners of the flannels. On every collection day, a cotton flannel without any chemical attractant was placed in the same area of the pasture to become an instrument to control the traps' capture efficiency. After 1 h, the white flannels were collected and placed in plastic bags for later identification and counting of the ticks. A total of 2,133 nymphs of Amblyomma sp., 328 adults of A. cajennense, and 292 adults of A. dubitatum were collected. Out of this total, the dry ice traps captured 1,087 nymphs (51 %), 188 A. cajennense (58.2 %), and 151 A. dubitatum (53 %), while the chemical traps captured 1,016 nymphs (47.6 %), 133 A. cajennense (41 %), and 133 A. dubitatum (46.5 %); 30 nymphs (1.4 %), 7 A. cajennense (0.8 %), and 8 A. dubitatum (0.5 %) were found on the control flannel. The capture potentials of ticks, nymphs, and adults, by the two models of traps tested, were statistically similar (p > 0.05). These results confirm the efficiency of the chemical trap enabling its use in areas of either difficult access or too distant from a dry ice supplier as is the case of forest areas where studies about ixodological fauna are generally carried out.

Spectroscopic evaluation of thymol dissolved by different methods and influence on acaricidal activity against larvae of Rhipicephalus microplus (Acari: Ixodidae).

The acaricidal activity of three thymol formulations was investigated at five concentrations (1.25, 2.5, 5.0, 7.5, and 10.0 mg/ml) on Rhipicephalus microplus larvae, and the behavior of its solubility in these formulations was analyzed. The thymol was dissolved in distilled water plus 1 % dimethylsulfoxide as adjuvant under two heating regimes (water bath in formulation 1 and hot plate in formulation 2) as well as without heating in 50 % ethanol and 50 % water (v/v). The acaricidal activity was assessed by the modified larval packet test, and the solubilization behavior was investigated by ultraviolet-visible spectroscopy, based on the Beer-Lambert law. With formulations 1 and 2, the mortality was greater than 95 % starting at the thymol concentrations of 5.0 and 7.5 mg/ml, respectively, while with formulation 3, this mortality level was reached starting at a concentration of 2.5 mg/ml, showing that the addition of ethanol in the solution enhanced the acaricidal action of thymol. This result was supported by the LC 90 values, which were 3.3, 2.4, and 1.6 mg/ml of thymol for formulations 1, 2, and 3, respectively. This result is related to the better solubility of thymol in the hydroethanolic formulation, since the spectroscopic analysis revealed that the thymol dissolved more completely in this formulation. This fact was evident once the R (2) obtained from the linear regression analysis of the relation absorbance × concentration of the formulations 1, 2, and 3 approached the optimal value (R (2) = 1) in the following sequence: 1, 2, and 3 (0.717, 0.901, and 0.968, respectively).

Acaricidal activity of the essential oils from Eucalyptus citriodora and Cymbopogon nardus on larvae of Amblyomma cajennense (Acari: Ixodidae) and Anocentor nitens (Acari: Ixodidae).

The present study evaluated the acaricidal activity of essential oils from Eucalyptus citriodora and Cymbopogon nardus on non-engorged larvae of Amblyomma cajennense and Anocentor nitens. In order to carry out the study, six groups were formed, each concentration being a treatment (6.25%, 12.5%, 25%, and 50%, respectively) and also with the creation of a control group (distilled water) and a positive control (Deltametrine). For each treatment, approximately 100 larvae of these ticks were placed onto filter papers (2 x 2 cm) impregnated with the concentrations used to test. Next, the envelopes were closed bearing inside the filter paper with measurements of 6 x 6 cm. For each group, six repetitions were performed, and after 24 h live and dead larvae were counted. This procedure was carried out for two essential oils on the two species of ticks. For A. cajennense, the acaricide efficacy of E. citriodora oil was of 10.8%, 35.3%, 34.5%, and 53.1%, whereas the efficacy of C. nardus was of 0.0%, 0.0%, 0.0%, and 61.1% at concentrations of 6.25%, 12.5%, 25.0%, and 50.0%, respectively. In relation to A. nitens, the acaricide efficacy of E. citriodora oil was of 20.1%, 84.5%, 89.2%, and 100.0%, whereas the efficacy of C. nardus was of 0.0%, 90.8%, 100.0%, and 100.0% at concentrations of 6.25%, 12.5%, 25.0%, and 50.0%, respectively. The results indicate that the essential oils tested showed a promising acaricidal activity mainly on A. nitens larvae.