Association between Duddingtonia flagrans, dimethylsulfoxide and ivermectin for the control of Rhabditis spp. in cattle

Cattle parasitic otitis caused by the nematode Rhabditis spp. is a serious health problem in Brazil, a situation which is confounded by lack of effective control measures. In vitro studies associating biological and chemical control as an alternative method showed promising results. The objective was to evaluate the combined use of Duddingtonia flagrans (AC001), 10% dimethylsulfoxide (DMSO), and 1.9% ivermectin for the in vivo control of Rhabditis spp., in naturally infected Gyr cattle. For this purpose, 48 animals, whose infection in both ears was diagnosed, were randomly assigned to 6 groups: group 1 (ivermectin 1.9%); group 2 (10% DMSO); group 3 (AC001); group 4 (ivermectin 1.9% + 10% DMSO w/v); group 5 (1.9% ivermectin + AC001 w/v); group 6 (10% DMSO + AC001 v/v). The treatments were performed in a single dose, in the right ears, with the left ears remaining untreated, as a control group. There was a significant reduction (p < 0.01) in the number of nematodes in the treated groups in relation to the control, with the following best efficacies: groups 1 and 2, 47% and 52.9%, respectively, 7 days after treatment; groups 3, 4, and 5, 47.8%, 48.6% and 36.7%, respectively, 14 days post-treatment; group 6, 38.4%, 21 days post-treatment. It was concluded that the combination of chemical compounds and D. flagrans in a single application was effective for the in vivo control of Rhabditis spp. in naturally infected cattle.

Rhabditis spp. transmission is facilitated in the presence of dipterans (flies), leading to infections predominantly in both ears, which may progress to abundant purulent secretion with a large number of parasites (Vieira et al., 2001;Verocai et al., 2007).
Specifically in relation to treatments used in parasitic otitis, there are many drugs that have been used; however, the results are unsatisfactory and the recurrence of infections is a routine problem in animals (Verocai et al. 2007; Barbosa et al., 2016). Thus, alternatives that may be synergistic to control with anthelmintic drugs have been researched. Recently, Sobral et al. (2019) demonstrated the nematophagous fungi in vitro effectiveness, including Duddingtonia flagrans, in the control of Rhabditis spp. In another recent study, Ferraz et al. (2019) report that the use of 1% dimethylsulfoxide (DMSO) and 100% mineral oil together with nematophagous fungi were effective in vitro in the destruction of Rhabditis spp.
There are no reports on the association of nematophagous fungi with chemical compounds for the in vivo control To verify the infection by Rhabditis spp., 7 days before beginning the experiment, the animals with signs of external otitis were restrained in a containment trunk and their head was immobilized. Subsequently, the technique of ear canal washing was performed. Fifty milliliters of saline solution was applied in both ear canals. Subsequently, the material was collected in a sterile stainless steel tray and placed in 50 mL Falcon tubes. Then, the samples were packaged and sent for laboratory analysis. After confirming the presence of infection by Rhabditis spp. in both ear canals, by visualization under an optical microscope, 10 × and 40 × objectives, the animals were able to the experiment.
Six groups with eight animals each were assembled. Experimental protocols were performed only on the right ears, on day 0 (D0): group 1 -ivermectin 1.9%, 500 µl/ animal, topically in the ear canal; group 2 -ear canal washing with a 10% DMSO solution (45 mL/animal); group 3 -12 mL solution containing 3 × 10 6 D. flagrans conidia in mineral oil vehicle, topically in the ear canal; group 4ivermectin 1.9%, 500 µg/animal + ear canal washing with a solution of 10% DMSO (45 mL/animal) w/v, both topically in the ear canal; group 5 -Ivermectin 1.9%, 500 µg/animal, topically in the ear canal + 12 mL solution containing 3 × 10 6 D. flagrans conidia in mineral oil vehicle w/v; group 6 -ear canal washing with a 10% DMSO solution (45 mL/ animal) + 12 mL solution containing 3 × 10 6 D. flagrans conidia in mineral oil vehicle v/v. In all animals, the left ears served as the control group, in which only the washing was performed on D0 with 50 mL of saline solution.
The percentage of reduction/efficacy of the experimental protocols in the studied groups, in relation to the control, was evaluated using the following formula: The results obtained were analyzed by means of analysis of variance (ANOVA) at a 5% probability level. The Tukey's test was applied as a post-assay test.
There was a significant reduction (p < 0.01) in the number of nematodes in the treated groups compared to the control group (Table 1). Groups 1 and 2 showed higher reductions of nematodes seven days after treatments (D7), 47% and 52.4%, respectively. Groups 3, 4, and 5 showed higher reductions in infections 14 days after treatments (D14), with 47.8%, 48.6%, and 36.7%, respectively. Group 6, on the other hand, showed higher reductions 21 days after treatments (D21), with 38.4%.
The authors emphasize the fact that the Gyr herd and its crossbreeds are important in milk production in Brazil, and therefore the parasitic otitis caused by Rhabditis spp. causes incalculable damage to this livestock activity (Bossi et al., 2015;Sobral et al., 2019). The literature argues that  Table 1 Averages and percentages of reduction, R(%) of the nematode Rhabditis spp., recovered in the groups of treated animals: group 1 -ivermectin 1.9%, 500 µg/animal, topically in the ear canal; group 2 -ear canal washing with a 10% DMSO solution (45 mL/animal); group 3 -12 mL solution containing 3 × 10 6 D. flagrans conidia in mineral oil vehicle, topically in the ear canal; group 4 -ivermectin 1.9%, 500 µg/animal, topically in the ear canal + ear canal washing with a solution of DMSO 10% (45 mL/animal) w/v; group 5 -iver-mectin 1.9%, 500 µg/animal, topically in the ear canal + 12 mL solution containing 3 × 10 6 D. flagrans conidia in mineral oil vehicle w/v; group 6 -ear canal washing with a 10% DMSO solution (45 mL/ animal) + 12 mL solution containing 3 × 10. 6 D. flagrans conidia in mineral oil vehicle v/v; over days 7 (D7), 14 (D14), and 21 (D21), in relation to their respective control groups before treatment, at day 0 (D0) Averages followed by the same lowercase letter in the lines do not differ statistically (p ≥ 0.01) by Tukey's test the control of parasitic otitis is difficult, due to a large variety of therapeutic protocols tested without success (Souza et al., 2008;Leite et al., 2013).
In the present study, the fungus D. flagrans (AC001) was applied topically in a mineral oil vehicle (group 3), showing a maximum reduction of 47.8%, 14 days after treatment. It is important to note that this is the first report of in vivo/topical administration of D. flagrans in the control of Rhabditis spp. under natural conditions, which further reinforces the use of this fungus in parasite control. Rodrigues et al. (2021) recorded the efficacy and safety of the commercial formulation Bioverm® (D. flagrans) and which in the future may act in the prevention and control of parasitic otitis. This result is still in agreement with what was proposed by Ferraz et al. (2019), who associated nematophagous fungi with chemical compounds in an in vitro assay and obtained promising results, with a future premise of designs under natural conditions, as presented in this paper. It is noteworthy, however, that other vehicles, forms of application, and complementary dosages of treatments can contribute to a better effect in the elimination of nematodes. Thus, it is important to continue with this research line.
In groups 5 and 6, D. flagrans was associated with the chemical compounds, ivermectin 1.9% and 10% DMSO, respectively, with reduction, after 21 days, of 32.6% and 38.4%. We chose to use ivermectin 1.9% in the form of a topical paste to provide better adherence of the product to the animals' ear canal. Even so, when the anthelmintic was used alone, in group 1, a reduction of 47% (D7) was observed, demonstrating a low effect on the elimination of nematodes. Because it is widely used as an endectocidal drug, the incorrect use of ivermectin over the decades has resulted in parasite resistance, a serious and global problem (Laing et al., 2017). Other unsatisfactory results with the use of ivermectin 0.5% have already been reported (Verocai et al., 2009;Barbosa et al., 2016).
In group 2, the use of a 10% DMSO solution showed a reduction of 52.4%, 7 days after treatment. However, the combination of the compound with other drugs or the formulation of a paste with better fixation may be the solution for more satisfactory results in the future. Dimethylsulfoxide is an organic compound from the processing of petroleum used in the pharmaceutical industry. Studies point to various activities for this compound, such as anti-inflammatory, antioxidant, rapid permeability in tissues, analgesic, immunomodulator, and substance carrier (Crivellenti et al., 2013;Picoli et al., 2015).
It was concluded that the combination of D. flagrans, 10% DMSO, and 1.9% ivermectin was effective for the in vivo control of Rhabditis spp., which is an important contribution to the control of cattle parasitic otitis.