Experimentally confirmed toltrazuril resistance in a field isolate of Cystoisospora suis

Coccidiosis is a major parasitic disease affecting a wide range of livestock and wild animals globally. In pigs, Cystoisospora suis (syn. Isospora suis) is the most pathogenic species of swine coccidian and most severely affects suckling piglets [1–4]. Clinical signs include pasty to watery non-hemorrhagic diarrhea, weight loss and ill thrift [5–10]. At present, cystoisosporosis is considered as one of the leading causes of diarrhea in neonatal piglets with high prevalences worldwide [3, 7, 11–14]. Oocysts are highly resistant to desiccation and antimicrobial compounds [14, 15] making elimination virtually impossible once they have been introduced into the farm. The disease shows a very high morbidity with low mortality, and not all the piglets in a litter are equally affected, resulting in reduced, uneven weaning weights and thus, ultimately in often significant economic losses [5, 11, 16, 17].

In the European Union (EU), control of cystoisosporosis is commonly achieved with a single oral administration of toltrazuril in the prepatent period (day 3–5 of life). Baycox® is the trade name of a broad spectrum anticoccidial drug containing the triazine trione toltrazuril [18]. Use of Baycox® in piglet coccidiosis was first authorized in Australia in 1998 to be administered once in the first week of life at a dose of 20 mg/kg body weight (BW) [19]. A single oral treatment with toltrazuril administered during the prepatent period provided effective and sustained suppression of oocyst shedding and diarrhea in piglets experimentally infected with C. suis [17, 20–22] and under field conditions [23–28]. However, emerging resistance in poultry coccidia against anticoccidials including toltrazuril is of growing concern [29–31]. Constant treatment regimens with toltrazuril have been applied for controlling porcine cystoisosporosis in the EU for almost two decades now, but the possibility of resistance development in C. suis isolates has not been addressed so far, despite limited availability of treatment alternatives. In 2014, a pig farmer in The Netherlands, with a farrow-to-finish herd of 330 sows, complained about pasty feces in 60% of the piglets from 10 days of age until weaning despite treatment with the recommended dose of toltrazuril. The involvement of bacterial and viral pathogens was excluded as the possible pathogens associated with piglets’ diarrhea could neither be re-isolated nor be detected by polymerase chain reaction (PCR). Presence of villous atrophy and fusion in the histological sections of jejunum indicated cystoisosporosis [32, 33], which was later confirmed by the detection and isolation of C. suis oocysts in the fecal samples. Evaluation of the administered amount of toltrazuril on the farm level revealed no under-dosing. Moreover, application of twice the recommended dose (40 mg/kg) of toltrazuril also did not have any effect on the clinical picture and thus loss of efficacy was suspected. In the present study, the efficacy of toltrazuril against C. suis infections in suckling piglets was evaluated in experimental infections with the mentioned field isolate, Holland-I, and a toltrazuril-sensitive strain, Wien-I. In the past, experimental studies have indicated development of resistance under field conditions in Eimeria of poultry [34–36]. To our knowledge, this is the first report of experimentally confirmed toltrazuril resistance in a field isolate of C. suis.

Resistance to all classes of anticoccidials is well described in avian coccidia [30, 38, 39]. Despite the long-term use of toltrazuril in piglet production and the lack of satisfying treatment alternatives, the possibility of resistance development has not been addressed so far. In the present study we evaluated the efficacy of toltrazuril against a C. suis isolate from a Dutch farm complaining about symptoms typical of coccidiosis despite metaphylactic treatment with the recommended dose of toltrazuril. In an experimental setup, two litters were infected with the isolate in question and treated with 0, 20 or 30 mg/kg BW of toltrazuril. Additionally, a third litter was infected with a toltrazuril-sensitive strain (Wien-I) and treated with 20 mg/kg BW of toltrazuril or sham treated to confirm effectiveness of the Baycox® batch used. Due to animal welfare reasons only a minimum number of animals were infected with this strain; therefore, statistics could not be employed for these groups. However, the positive effect of toltrazuril on oocyst excretion, fecal score and body weight gain was sufficiently shown in several earlier field and experimental studies [7, 17, 20, 21, 24, 26, 40, 41]. It completely inhibits the development of all parasitic stages of C. suis and, given during the prepatent period of infection, prevents tissue damage and consequently the emergence of diarrhea [42].

Resistance is described by the World Health Organization [43] as the “ability of a parasite strain to survive and/or multiply despite the administration and absorption of a drug given in doses equal to or higher than those usually recommended but within tolerance of the subject”. This definition can also be employed for coccidia. The reliability of toltrazuril to significantly reduce the excretion of C. suis oocysts was already shown in a number of experimental studies [20, 21, 41, 42]. Mundt et al. [7] described a complete suppression of oocyst shedding in experimentally infected and toltrazuril-treated animals while every untreated animal shed parasites at least once. In another study conducted by Mundt et al. [17], the treatment with toltrazuril resulted in significantly fewer mean excretion days (0.6 vs 4.0), fewer piglets with oocyst excretion (30 vs 91%) and a significantly lower mean OpG (144 vs 17,797) 5 to 11 days post-infection (dpi). This is in accordance to a field study conducted by Kreiner et al. [24] where the number of C. suis positive samples was significantly lower in toltrazuril-treated compared to untreated piglets in different herds. The same effect was observed in this study for group Wien-20, where oocyst shedding was completely suppressed by the treatment. By contrast, treatment with toltrazuril did not impair oocyst development and excretion in the groups infected with the Dutch isolate, Holland-I. In avian coccidiosis complete drug resistance is defined by ineffectiveness despite higher doses [44]. Every single piglet infected with the Holland-I isolate shed oocysts regardless of the toltrazuril dose. Moreover, the number of AF countable excretion days, the AUC for OpG, the maximum individual OpG as well as the mean OpG from SD 9 to 19 was comparable between the control and treatment groups of this isolate, indicating complete drug resistance. The above mentioned parameters tended to be higher in groups Holl-Ctrl, Holl-20 and Holl-30 compared to group Wien-Ctrl, and the prepatent period for excretion detected by AF technique was shorter in Holland-I compared to Wien-I. Variations in prepatent periods can be attributed not only to factors such as infection dose or age and health of piglets (which were comparable between all groups) but also to the virulence of C. suis isolates [7, 45, 46]. However, as oocyst excretion underlies large individual variations [7, 47–49] reliable conclusions about possible differences between strains regarding this particular trait cannot be drawn at this point in time.

While mean fecal consistencies remained below 1.2 in group Wien-20, all other groups developed increased mean fecal scores after infection, indicating enteritis as a consequence of parasite replication [50]. Diarrhea was observed in all groups except group Wien-20, but prevalences and numbers of diarrhea days were generally low. It has been previously shown that toltrazuril successfully reduces fecal scores and suppresses diarrhea in piglets infected with C. suis [17, 41, 42, 51]. In a field study, Scala et al. [26] found the overall diarrhea prevalence to be significantly lower in toltrazuril-treated animals compared to untreated animals. Similarly, Kreiner et al. [24] found treated animals to have a significantly lower mean FS and significantly fewer diarrheic fecal samples in the field. In two trials conducted by Joachim & Mundt [20] not a single piglet with diarrhea was observed after treatment with toltrazuril while almost all untreated animals had a FS of 3 or 4 (85.7 and 100%, respectively) at least once. This was also described by Mundt et al. [7] who observed an average FS of 2 or more in experimentally infected piglets throughout the study, with daily diarrhea prevalences between 25 and 75%. On the other hand, the mean FS of animals treated with toltrazuril remained between 1 and 2 during the entire sampling period. In this study, treatment had obviously no effect on fecal consistency of the piglets infected with the Dutch isolate. The group Holl-20 showed the highest mean fecal score, the highest prevalence of diarrhea and most diarrhea days among the three groups. These parameters were also similar in groups Holl-Ctrl and Holl-30, indicating complete lack of clinical efficacy of toltrazuril. Despite the limited data set available so far for Holland-I, this strain might be of low virulence since overall prevalences and days of diarrhea were low and a FS of 4 was not observed even once. However, inter-and intra-litter deviations cannot be excluded in this setting. Just as with oocyst excretion, the development and severity of diarrhea varies between litters and individuals [48, 52, 53] and is also influenced by other factors [7].

Chapman [54] considered an Eimeria strain to be resistant if the weight gain of treated infected chicken did not differ significantly from that of untreated infected controls. In fact, treatment with the recommended or the elevated dose did not have a significant effect on body weight gains in the Dutch isolate, indicating resistance to toltrazuril. This finding is not surprising as the mean fecal score, which was negatively correlated with the individual weight gain, did not differ significantly between the control and treatment groups of isolate Holland-I. It has previously been shown that diarrhea is negatively correlated with the body weight gain in C. suis infections and that the application of toltrazuril significantly increases the weight gain of piglets compared to untreated infected controls [7, 17, 26, 40], although for the groups infected with Wien-I this could not be shown due to the small group size.

On grounds of the clinical picture observed during the study, it can be assumed that the bacterial agents identified by microbiological examination are facultative pathogenic subtypes regularly found in porcine intestinal flora [55, 56]. This is underpinned by the fact that no sign of disease occurred in group Wien-20, despite the presence of the same bacteria as in all other groups. Jonach et al. [57] did not find statistical differences in the intestinal abundance of E. coli or C. perfringens when comparing piglets with and without diarrhea. This was confirmed by Ruiz et al. [58] who found similar prevalences in piglets with diarrhea and control piglets while frequencies of C. suis were significantly higher in diarrheic piglets.

To our knowledge, this study is the first to experimentally confirm toltrazuril resistance in a C. suis isolate. There are currently no satisfying treatment alternatives available [20, 23], underlining the need of new intervention strategies against porcine neonatal coccidiosis. Sulfonamides have been suggested against porcine coccidiosis but a short-term oral administration was shown to have an unsatisfying effect [17, 26]. Only an injectable sulfonamide, repeatedly administered for 6 to 7 days, had an effect on parasite development and clinical outcome comparable to that of toltrazuril under experimental conditions [20]. Such a treatment may be considered as an alternative in cases of toltrazuril resistance, but it is labor-intensive and unsuited for routine treatment. Therefore, alternative control strategies to chemometaphylaxis are desirable [59].

Luckily, it seems that resistance in C. suis develops far slower than in avian coccidia. Vertommen et al. [60] described the development of resistance to Baycox® against Eimeria on a broiler farm within four fattening periods. Nevertheless, the rise of further resistant C. suis isolates may just be a matter of time as the extensive use of a drug over a longer period of time will inevitably lead to decreased efficacy [29, 44]. Currently, the sensitivity of C. suis isolates to toltrazuril can only be evaluated in vivo. A less laborious and faster in vitro assay, possibly employing the already established cell culture system [61], would be advantageous.

Toltrazuril resistant C. suis isolates are a potential threat to pig farming as no other effective and economically sustainable alternative treatment is available. All piglets infected with the field isolate of C. suis Holland-I showed appreciable levels of diarrhea and oocyst excretion unresponsive to treatment. Therefore, veterinarians and farmers should be aware of the possibility of resistance development with long-term application of toltrazuril in intensive piglet production systems. In the absence of vaccines and effective anticoccidial agents other than toltrazuril, routine fecal screening and periodical assessment of efficacy of toltrazuril must be considered essential for the sustainable control of cystoisosporosis. In cases of reduced efficacy optimized hygiene measures employing regular chemical disinfection with a cresol-based product must be enforced.