Nematode-bacteria interactions in bovine parasitic otitis

Abstract

Bovine parasitic otitis poses challenges in diagnosis, treatment and involves various agents, such as bacteria, fungi, mites, and nematodes. This study focused on the nematodes and bacteria isolated from the auditory canals of dairy cattle. A total of twenty samples were collected from dairy cattle in two states of Brazil. The results showed that Metarhabditis freitasi and M. costai nematodes were identified in 75% of samples. Bacterial species from the ear, identified via mass spectrometry, revealed that different strains were present in 65% of the cattle. Mycoplasma spp. were identified in 45% of samples through molecular techniques. Gram-negative bacteria and Mycoplasma spp. were exclusively found in nematode-infected cattle. Furthermore, the bacteria exhibited resistance to multiple antimicrobial classes, and demonstrating multiresistance. Electron microscopy revealed biofilm aggregates on the cuticle of Metarhabditis spp., suggesting a potential role of these nematodes in bacterial migration and interaction with nervous tissue. Thirteen bacterial strains demonstrated biofilm formation ability, indicating their potential pathogenic role. This research highlights the persistent and complex nature of parasitic otitis, emphasizing the significant role of nematode-bacteria associations in its pathogenicity. The presence of resistant strains and biofilm formation underscores the challenges in managing the diagnosis and treatment of bovine parasitic otitis.

Figure 1. Nematodes were collected by inserting sterile swabs into the auditory canal of cattle, performing rotary movement, and immediately transferring them to sterile tubes containing 2 mL of PBS. To determine the parasite load, the tube contents were homogenized, and 500 µL aliquots were transferred to microtubes. The material was homogenized again, and 10 µL was immediately removed and analyzed in triplicate via bright-field light microscopy. The samples were fixed via immersion in Karnovsky solution.

Figure 2. For ultrastructural analyses, the fixed worms were washed in PBS, adhered to coverslips with 1% gelatin, fixed in 1% OsO₄ and 0.8% K₃[Fe(CN)₆], dehydrated in a graded ethanol series (30%-absolute), critical point dried in CO₂, mounted on stubs, coated with gold, and examined via a scanning electron microscope.

Figure 3. Bacterial collection and isolation were performed by inserting sterile swabs into the auditory canal of cattle and immediately transferring them to Stuart transport medium. Subsequently, various culture media plates were incubated at 35 ± 2 °C for 24 to 48 hours. Colonies that grew were selected for smear preparation and Gram staining to characterize their morphology and staining properties via bright-field light microscopy. Pure culture colonies were incubated in the same medium and stored in GC media supplemented with 20% glycerol at -20 °C.

Figure 4. For mycoplasma collection, the ear canals were washed with 50 mL of sterile PBS via a syringe coupled with a hose. The samples were then stored in microtubes and transported in an isothermal box stored in glycerol at -20 °C.

Figure 5. Confocal microscopy of Metarhabditis sp. A- General morphology of a male showing the esophageal bulb (b) and testis (t). B- Posterior region of the male, showing the spicule (arrow). C- Posterior end of the male showing the lateral view of the bursa and genital papillae (arrows). D- Posterior region of the female showing a larva (la), eggs (*), gonad (g), terminal region of the intestine (i), and anus aperture (a).

Figure 6. Number of Gram-positive and Gram-negative bacteria isolated from samples collected from the ear canal of bovines from Piraí (PI) and Castanhal (CA) farms. Gram-negative bacteria were isolated only from bovines infected with nematodes. Gram-positive bacteria were isolated from infected and noninfected cattle.

Figure 7. Scanning electron microscopy of bacteria adhered to the cuticle of Metarhabditis sp. A- Anterior end showing bacilli (b) and cocci (c) adhering to the oral opening. B- Dorsal view of the posterior region of a male completely covered by the biofilm-like structure. C-Details of the biofilm-like structure showing bacilli (b) and cocci (c) forming a dense packing of bacteria on the nematode cuticle. D- Ventral view of the male posterior region showing a biofilm-like structure. E- Details of cloacal opening with bacilli (b) and cocci (c). F- Tip of the posterior end showing adhered bacteria. G- Lateral view of the middle region of the female, showing the biofilm-like covering of the opening of the vulva. H- Details of the biofilm-like structure showing bacilli (b) and cocci (c) forming a dense packing of bacteria on the opening region of the nematode vulva.

Figure 8. Light microscopy (bright-field) images of biofilm formation experiments. A and B- Staphylococcus chromogenes (strain 7) forming a moderate biofilm. C- Escherichia coli (strain 3 - CLED 1) forming a poor biofilm. D- Bacillus pumilus (strain 6 - AS3) showing no biofilm formation.