2.1. Databases and Strategy of Search

For this review three databases were employed: PubMed (MEDLINE), Scopus and Web of Science. Three searches were performed to include the highest number of articles, with the following terms and Boolean connectors: (1) “Trichomonas gallinae” AND “Treatment”, (2) “Trichomonas gallinae” AND (“Metronidazole” OR “Nitroimidazole” OR “Carnidazole” OR “Dimetridazole” OR “Ronidazole” OR “Ornidazole” OR “Tinidazole”) AND (“Resistance” OR “Sensitivity”), and (3) “Trichomonas gallinae” AND (“Natural products” OR “Essential oils” OR “Natural compounds” OR “Plant products” OR “Plant extracts”). Articles with no restriction for the date of publishing were included. Additionally, a search within the cited references in each article was performed and related references included. The final search was carried out on 8 October 2022. Duplicates were removed from the list before adding references to Mendeley. The abstract of each article was manually reviewed and agreement about the inclusion of the articles in the systematic review was achieved by all the authors.

3.2. Treatments against T. gallinae

Treatment of avian oropharyngeal trichomonosis has improved since the first attempts made during the 1940s and 1950s. The employment of copper sulfate, hydrochloric acid, Lugol’s solution or mercuric chloride were tested on naturally infected pigeons with anti-trichomonal (AT) effect, but prolonged doses, poor palatability, or adverse effects were noticed in most cases [11,12] and they were soon dismissed. Other substances with slight or null AT effect tested in vitro or in natural or experimental infections were: 1. furazolidone, only efficient at high doses with toxic effects [13], 2. penicillin and streptomycin [12], 3. auremycin, nitrophenide and sulfamides [14], 4. Osmaron B [8], and 5. nihydrazone [15].

Several drugs have been tested with success and low toxicity in the therapy of avian trichomonosis and most of them are still employed when clinical cases appear. The majority of the studies were performed by employing naturally or experimentally infected domestic pigeons (Columba livia) (Table 1, Supplementary Table S1). Nitrothiazoles were introduced in the decade of 1950 to treat trichomonads and other protozoal infections, starting with enheptin (also known as ANT or 2-amino-5-nitrothiazole) [14], and later with similar molecules or derivates. Enheptin was first tested under several conditions, including several doses, in naturally and experimentally infected pigeons, and with asymptomatic and symptomatic birds [14]. High efficacy was achieved with doses from 18 to 45 mg/kg/day administered for seven days, with lower doses being less effective and higher doses being toxic. The drug was also proved efficient at 0.125% (equivalent to 20 mg/bird/day) in aqueous solution for 6 days, both in natural and experimental infections, although some old pigeons appeared refractory to the treatment [8]. In an experimental procedure employing the highly pathogenic Jones´ Barn strain, a dose of 30 mg/bird/day for 14 days was necessary to reach 100% AT efficacy, and at this time, differences in drug sensitivity were observed between studies and apparently depending on the strains infecting the birds or the chronicity of the infection [16].

Metronidazole and dimetridazole were introduced in the 1960s and comparisons with enheptin were made, both in pill format and within drinking water, the latter being the recommended administration system by the authors [17]. Both drugs were effective at the dose of 50 mg/kg (approximately 15 mg/bird/day) for five days in chronically infected pigeons, or at 0.05% in drinking water administered for 3–5 days to naturally or experimentally infected pigeons [15,18], although temporary ataxia was observed in 12% of the birds if dimetridazole was administered for 6 days [15]. More trials were performed with dimetridazole, one of them administered between 5–10 mg/bird/twice daily for 3 days, with good results and no toxicity reported in experimentally infected pigeons [19], while another reported no efficacy if administered for 1 day (25 mg/day oral) but high and perdurable efficacy (up to 1 month) when administering the same dose for five days [20]. Finally, the combination of metronidazole (40–60 mg/day, 5–6 days) with oxytetracycline was effective in not severely affected pigeons [21]. It seems that the infection does not interfere with the pharmacokinetics of metronidazole [22]. In summary, five days is the recommended administration schedule to achieve 100% efficacy and perdurable results, with doses varying from 15 to 60 mg/bird/day.

Ornidazole was evaluated in the 1980s in infected pigeons, and under several formats of administration [23]. Doses of 2.5–5 mg/bird for 3 days dissolved in water were effective for at least seven days after treatment while lower doses were not. If administered in solid state, doses from 10 to 40 mg/bird/day for 3 days showed optimum AT effect for at least 17 days, the highest dose being the one recommended by the authors. Carnidazole is usually administered in one dose (10 mg/kg) and displayed lower efficacy when compared with metronidazole at the same dose in naturally infected pigeons (87.5% vs. 100%) [24]. When employed in a clinical situation, 10 mg/bird was enough to avoid clinical manifestations in adult pigeons; however, 60% of the squabs died of trichomonosis at 5 mg/bird [25]. The authors argued that early diagnosis was lacking in this case and was critical for treatment success. Nifursol 50% has been evaluated against experimental infections with T. gallinae. Doses of 600–1200 mg/kg of food were enough to achieve results comparable with the administration of metronidazole in drinking water [26].

Besides domestic pigeons, other birds, wild or domestic, have been treated against naturally acquired oropharyngeal avian trichomonosis, with clinical manifestations, employing in most cases metronidazole or dimetridazole (Table 2). Several cases of wild birds are described in the literature. Debilitated bald eagles (Haliaeetus leucocephalus) with oropharyngeal lesions were successfully treated with dimetridazole at doses of 50 mg/kg or total doses of 375–500 mg/bird, and in all cases, 1 day of treatment was not enough to eliminate the infection, and the authors needed at least two or three doses separated by 1–8 days, depending on the situation, to eliminate the trichomonads [27,28]. Besides, one asymptomatic bald eagle was treated with 50 mg/kg/12 h of metronidazole for five days to eliminate the infection [29]. Additionally, a debilitated cinereous vulture (Aegypius monachus) with mild oral lesions was treated with metronidazole (50 mg/kg, five days), although the treatment needed to be repeated to eliminate the infection [30]. In these two last cases, Trichomonas gypaetinii, another oropharyngeal trichomonad, was the etiological agent. Metronidazole has been used to treat clinical cases of trichomonosis (lesions in orpharynx, sinuses and internal organs) in several species of falcons at 50 mg/kg/day for 5–7 days, although injectable dimetridazole (5%, 0.25 mL) was employed to treat infraorbital sinuses [3]. During an outbreak of oral trichomonosis in wood pigeons (Columba palumbus), the administration of dimetridazole at 200 ppm in the food of game bird feeders ended the mortality events [31].

Domestic animals other than pigeons have been treated with both drugs. Clinically affected budgerigars (Melopsittacus undulatus) have been treated with dimetridazole (0.05 mg/g bird for seven days) in drinking water [4] or metronidazole (30 mg/kg, ten days) [7]. Metronidazole was partially effective when administered at 200 mg/L of drinking water for five days to canaries with clinical signs of oral trichomonosis, and completely eliminated the infection at 200 mg/kg for five days [6]. Turkeys with clinical signs of trichomonosis and raised together with pigeons and hens also recovered from the disease with metronidazole in drinking water for seven to ten days [32].

Carnidazole has been less widely employed to treat avian oropharyngeal trichomonosis in birds other than pigeons (Table 2). House finches (Haemorhous mexicanus) were treated at 20 mg/kg/day for five days [5] with some debilitated birds dying of trichomonosis. Severely affected sparrowhawk chicks were treated employing carnidazole, but only 2/5 recovered from the infection [9]. Carnidazole was also tried at several doses in naturally infected pink pigeons (Nesoenas mayeri) [33]. The employment of 10 mg/bird in one dose or 5–10 mg/ bird every 2–4 days until clinical signs ceased seemed effective in short term, although the infection eventually returns. Ronidazole and dimetridazole were also tested in drinking water, but variable attendance at drinking station was observed by the authors, probably not reaching the appropriate dose [33]. It seems that the low palatability of some drugs, the hierarchy of pigeons, the problems to dissolve some drugs, or the time of the year could interfere with the efficacy of the treatments, and these facts should be considered when establishing a treatment protocol .

Severely affected young birds are more prone to die of trichomonosis even if treated at the appropriate dose, and this fact has been observed in squabs [25], but also in canaries [6] and raptor chicks [9]. Highly debilitated birds of other species, such as budgerigars [4], pigeons, or house finches also died in spite of treatment administration. Prompt treatment seems to be crucial for the success of the therapy.

3.3. Resistance of T. gallinae to Nitroimidazoles

Nitroimidazole drugs are the treatment of choice against avian oropharyngeal trichomonosis. However, failures in the success of treatments in birds infected by T. gallinae were first reported in 1990 [34]. Since then, nine studies had been conducted specifically to demonstrate the existence of resistant strains to these compounds (Supplementary Table S2). Experiments had been carried out using metronidazole, dimetridazole, ronidazole, carnidazole, ornidazole and tinidazole. These studies showed that there are resistant strains of T. gallinae to all the nitroimidazole drugs used in birds . However, there are not studies directed to elucidate the mechanisms by which T. gallinae acquires resistance to nitroimidazole drugs. It has been assumed that the molecular mechanisms of resistance could be similar to T. vaginalis, since they are closely related. Some authors suggested that direct contact between the ingested drug and trophozoites within the lumen of the oral cavity or crop is required for complete protozoocidal activity [36]. Nitroimidazoles are frequently administered in drinking water, passing directly to the proventriculus in absence of crop content. The administration of these drugs with food would enhance the contact of the drug with the parasite, since food reduces the time to empty the crop, improving the response to the treatment .