Abstract
The efficacy of the anticoccidial drugs amprolium, clopidol, diclazuril, monensin, monensin + nicarbazin, narasin, narasin + nicarbazin, and salinomycin against field isolates of Eimeria acervulina obtained from a commercial broiler enterprise before and after immunization with a coccidiosis vaccine was investigated. Evaluated by weight gain, feed conversion, and lesion score following challenge, the isolate obtained before vaccination was resistant to all the drugs tested. By contrast, after vaccination the isolate was sensitive to all drugs evaluated by weight gain, and to most drugs judged by feed conversion and lesion score. It is concluded that vaccination had resulted in the restoration of sensitivity to these drugs.
1. Introduction
For many years, control of coccidiosis in broiler chickens has depended upon prevention or prophylaxis, involving the inclusion of anticoccidial drugs in the feed. A consequence of this has been the widespread acquisition of resistance by Eimeria spp., that has been documented for all drugs currently approved by regulatory authorities (Chapman, 1997). An alternative to medication is the vaccination of young chickens using live oocysts of Eimeria, the causative agents of coccidiosis (Chapman, 2000; Chapman et al., 2002; Williams, 2002).
An advantage of certain vaccines is that in addition to initiating an immune response that can confer protection against the pathological consequences of infection with the parasite, their use can result in the subsequent restoration of sensitivity to drugs. Restoration of sensitivity is considered due to the replacement of existing drug-resistant field strains with vaccinal strains that are drug-sensitive, the consequence of which is that in subsequent flocks, drugs are better able to control Eimeria infections (Chapman and Jeffers, 2014). This has been demonstrated for a few drugs, including the polyether ionophores monensin and salinomycin that are the mainstay of coccidiosis control in commercial broiler chickens, and the synthetic drug diclazuril (Chapman, 1994; Chapman and Jeffers, 2014; Chapman and Jeffers, 2015; Jenkins et al., 2010; Mathis and Broussard, 2006).
In this study we investigated whether use of a coccidiosis vaccine in a commercial situation can result in the restoration of sensitivity to a range of drugs currently available for the control of coccidiosis.
2. Materials and methods
2.1 Vaccine
The vaccine employed at the broiler farm from which the parasites were obtained was ADVENT® (Huvepharma Inc.) that comprises live oocysts of three species of Eimeria. It had been administered to newly hatched chicks in the hatchery via a spray cabinet according to the manufacturer's instructions to provide oocysts of E. acervulina, E. maxima, and E. tenella.
2.2 Birds and husbandry
Male Ross 308 broiler chickens, from the same parent flock, were obtained from a hatchery where they had been spray vaccinated against Infectious Bronchitis and Newcastle disease. They were transferred to a poultry facility in Belgium that had been thoroughly cleaned and disinfected, housed in battery cages on wire floors and provided with unmedicated feed and water ad libitum.
On day 14 of the study, 570 birds were weighed and randomly allocated to one of 114 cages (5 birds/cage) arranged in three rows. Only healthy birds within the body weight range of the estimated average (±10%) were included in the study. Cages measured 0.52 m2 and had a 75 cm feed trough and two drinking nipples to provide potable water ad libitum. Cages were constructed in accordance with EU Directive 86/609/EEC and EU Guideline L197 "for the accommodation and care of animals used for experimental and other scientific purposes" and were placed to provide similar lighting and environmental conditions.
2.3 Drugs and parasites
The drugs tested and the concentrations employed in the feed were:
- amprolium 125 ppm (AMP, Coxam®)
- clopidol 125 ppm (CLO, Coyden® 25%)
- diclazuril 2 ppm (DIC, Coxiril® 0.2%)
- monensin 100 ppm (MON, Coxidin®)
- monensin + nicarbazin 40 + 40 ppm (MON + NIC, Monimax®)
- narasin 60 ppm (NAR, Monteban®)
- narasin + nicarbazin 40 + 40 ppm (NAR + NIC, Maxiban®)
- salinomycin 60 ppm (SAL, Sacox® 120)
Samples of feed used were analyzed and all fell within 80-120% of their intended concentrations. Medicated feed was provided when birds were 14-22 days of age.
Parasites used in this study were obtained from fecal samples collected in five different farms from a large broiler integration in Russia when birds were 3-5 weeks of age.
In the year prior to vaccination, birds had received various anticoccidial programs in which CLO, decoquinate, NAR + NIC or robenidine were used in starter feeds and lasalocid, SAL, NAR, and maduramicin in grower feeds. Each fecal sample comprised a pool of nine sub-samples collected from random locations through the house. Each of the nine sub-samples included three fresh fecal droppings, one of which was a caecal dropping. Samples were collected in plastic pots containing potassium dichromate and combined for further processing in the laboratory. Fecal samples were first collected from a flock that had been medicated with robenidine in the starter feed and SAL in the grower feed. These samples are referred to as "before vaccination". The subsequent four flocks were not medicated with anticoccidial drugs by were vaccinated with the live coccidiosis vaccine. Following the vaccinated flocks, the subsequent flock was given SAL in both starter and grower feeds. During this flock, when birds were 3 weeks of age, fecal samples were collected as described above. These samples are referred to as "after vaccination". Oocysts were isolated and sporulated according to standard procedures (Shirley, 1995).
2.4 Experimental infection
Susceptible birds that had been reared in the absence of infection were inoculated with the isolates to provide sufficient oocysts for experimentation. Feces from these birds were collected from day 4-9 post inoculation, oocysts harvested, sporulated and stored in potassium dichromate. Thus, the parasites had been propagated once in birds prior to their use in the drug sensitivity evaluation.
Parasites obtained from the flocks before and after vaccination principally comprised E. acervulina, that were identified by their small size (18.3 x 14.6 μm) and location of characteristic lesions in the upper intestine (Long et al., 1976), but small numbers of E. tenella, E. maxima, and E. mitis were also detected. No attempts were made to purify the isolate.
A titration study was initially conducted in susceptible birds (eight birds/dose with four doses of increasing magnitude) to determine the number of oocysts to be administered in the drug sensitivity study. The objective was to determine a dose that would cause moderate pathological indices and intestinal lesions similar to those that can occur in field infections (such as a score of 2.0 on the scale devised by Johnson and Reid (1970)). Based upon this titration, the number of oocysts of E. acervulina given per bird in the sensitivity study was 1.38 x 105 for the isolate obtained before vaccination (referred to as "isolate before vaccination") and 1.05 x 105 oocysts for the isolate obtained after vaccination (referred to as "isolate after vaccination").
2.5 Experimental design for the test of drug sensitivity
The experimental design is shown in Table 1. There were 19 treatments that were randomized withing six blocks.
Treatment 1 was an uninfected unmedicated control (UUC). Treatments 2 and 11 were infected with the parasites obtained before and after vaccination respectively and unmedicated (IUC). Treatments 3-10 and 12-19 were infected and supplemented with drugs as indicated in Table 1.
Each of the treatments comprised a cage containing five birds that was replicated six times thus providing 30 birds/treatment. Birds were randomly assigned to treatment groups when they were 14 days of age and given either unmedicated or medicated feed as indicated in Table 1. Two days later (day 16), those to be infected were orally inoculated with the parasites in 1 ml of water. The UUC were sham inoculated. Six days later (day 22), all birds were euthanized, and their intestines removed for lesion scoring as described by Johnson and Reid (1970).
Bodyweight was recorded on days 14 and 22, the gain in weight from d14-22 calculated, and the daily weight gain determined.
Feed consumption was recorded from days 14-22 and feed conversion calculated.
Any mortality was recorded, and birds were necropsied by a qualified veterinarian to establish whether coccidiosis was the cause of death.
Criteria recorded for determination of drug efficacy included daily weight gain (DWG), feed intake (FI), feed conversion (FCR), and lesion score (LS).
2.6 Statistical analysis
Continuous outcome variables, which are DWG and FCR were analyzed with a linear model, with the independent variables being the different treatments and the inoculum (taken before or after vaccination). The DWG was recorded by weighing individual birds. Since they are housed in cages, birds belonging to the same cage are not completely independent. This was taken into account by including a random factor. For FCR, estimates were for the entire cage and hence, a fixed effect linear model was applied.
The reference was taken to be the IUC and results are presented as the mean difference compared with this treatment. Ordinal outcome variables (lesion scores) were analyzed with a proportional odds model. This model takes into account the ordinal nature of the lesion scoring system and evaluates whether there is an association between the independent variables i.e., the treatments and the probability of falling into a higher lesion score category. The IUC was set as the reference level. All analyses were performed with the R software version 3.6.5. The α-level for significant differences was set at 0.01.
3. Results
3.1 Mortality
Mortality in the IUC and medicated birds given the isolates before and after vaccination was 2.6 and 6.3% respectively. No deaths could be attributed to coccidiosis and mortality was therefore not analyzed further.
3.2 Weight gain
DWG of birds given the different treatments is presented in Table 2.
The DWG of the IUC birds was significantly lower than that of the UUC whether they were infected with isolates obtained before or after vaccine use. There were no significant differences between the IUC and medicated birds given the isolate obtained before vaccination, indicating that none of the drugs were able to control the infection. By contrast, where birds were infected with the isolate obtained after vaccination, the DWG of the medicated birds were significantly greater than the IUC indicating that all the drugs were effective.
3.3 Feed intake
There were no significant differences in FI between treatments (data not presented).
3.4 Feed conversion
The FCR of the IUC birds was greater than that of the UUC whether they were infected with isolates obtained before or after (p<0.01) vaccine use (Table 3).
FCRs of the medicated birds given the isolate obtained before vaccination were not significantly different than the IUC indicating that none of the drugs were effective. However, birds given CLO, DIC, MON + NIC, NAR + NIC, and SAL that were infected with the isolate obtained after vaccination had a significantly lower FCR than the IUC, indicating that the drugs were able to control the infection. The FCR of birds given AMP, MON, and NAR was not significantly different from the IUC indicating no improvement in FCR.
3.5 Lesion score
Lesions in the upper intestine (duodenum), attributed to E. acervulina, are shown in Table 4.
Scores from medicated birds infected with the isolate obtained before vaccination were not significantly different (and in the case of SAL significantly higher) than the IUC, thus indicating that the drugs were unable to control this isolate. Scores from birds given DIC, MON, MON + NIC, NAR + NIC and SAL that were infected with the isolate obtained after vaccination were significantly lower than the IUC indicating they were able to control the infection. AMP, NAR and CLO however, were ineffective in preventing lesions.
Few lesions were detected in the mid-intestine and caeca of the IUC birds. These low scores were likely due to the small numbers of E. maxima and E. tenella in the field isolate and therefore the data for these regions of the intestine were not analyzed.
4. Discussion
In this study, a commercial broiler enterprise in Russia practiced rotation programs for the control of coccidiosis that comprised medication with various anticoccidial drugs. Details were not available, but included use of synthetic compounds CLO, decoquinate, NIC, and robenidine, and the ionophores lasalocid, maduramicin, NAR and SAL.
This was followed by four successive flocks in which birds were given the live coccidiosis vaccine. In the subsequent flock after vaccination, birds were medicated with SAL in the starter and grower feeds. By collecting oocysts from fecal samples obtained before and after vaccination, it was possible to determine whether this program resulted in a change in the sensitivity of the parasites to a range of drugs commonly employed to control the disease.
The isolates obtained before and after vaccination were identified as primarily E. acervulina, judged by the small size of the oocysts and the lesions observed following infection of susceptible birds.
Numerous studies have reported that E. acervulina is the most common species of Eimeria encountered in commercial broilers although other species are frequently present (e.g., Haug et al., 2008; Jeffers, 1974; Jenkins et al., 2017; Williams et al., 1996). Many studies have also shown that resistance to anticoccidial drugs is widespread in field isolates of E. acervulina and other species of Eimeria (reviewed by Chapman, 1982; Chapman, 1997; Jeffers, 1989; Peek and Landman, 2003).
Experimentally, it has been shown that it is possible for a strain of Eimeria to become resistant to several drugs (Chapman, 1984), a phenomenon known as multiple resistance, and indeed such strains have been reported from field isolates of Eimeria, especially for E. acervulina which has a high reproductive index and therefore a propensity for rapid resistance development (Jeffers, 1974; Ryley, 1980).
In the present study, using the criteria of DWG, FCR, and LS, the isolate obtained before vaccination was not controlled by any of the drugs tested including the synthetic drugs AMP, CLO and DIC, the ionophores MON, NAR and SAL, and the combination anticoccidials MON + NIC and NAR + NIC. A comprehensive record of all drugs used at the broiler farm was unavailable, but it is likely that coccidia may have been exposed to many of these drugs thus resulting in multiple resistance.
Evaluated by DWG, all drugs tested were able to control the field isolate given after vaccination. With the exception of AMP, MON and NAR, all drugs were effective judged by FCR, and with the exception of AMP, NAR and CLO all drugs were effective judged by LS. A summary of these findings is shown in Table 5 and clearly indicate that whereas drugs were ineffective prior to vaccination, after vaccination efficacy was improved suggesting that drug sensitivity had been restored.
Restoration of drug sensitivity in broilers has previously been demonstrated for MON, SAL and DIC following use of a live non-attenuated vaccine (Coccivac-B®; Chapman, 1994; Chapman and Jeffers, 2015; Jenkins et al., 2010; Mathis and Broussard, 2006), and partial restoration to MON and DIC after use of an attenuated vaccine (Paracox®-5; Peek and Landman, 2006). This study extends these observations to include other drugs frequently used to control coccidiosis and a third live coccidiosis vaccine.
An explanation provided for this phenomenon is that strains of vaccinal origin replace existing drug-resistant strains in a broiler house (Chapman and Jeffers, 2014). The component strains of bothe Coccivac-B® and Paracox®-5 were originally isolated before most drugs were introduced and they have been maintained in laboratory conditions ever since, consequently they have never been subject to the selective drug pressures imposed upon field strains and are inherently sensitive to drugs. The sensitivity of ADVENT® to monensin and salinomycin had been already demonstrated (unpublished data), the result of this study suggests that strains present in the vaccine may also be sensitive to the other tested drugs.
Thus, vaccination with such strains may repopulate poultry houses with sensitive strains and this will result in restoration of drug efficacy apparent if chemotherapy is resumed. Loss of resistance following admixture of sensitive and resistant strains has been reported in laboratory experiments (Ball, 1966; Jeffers, 1976; Long et al., 1985; McLoughlin and Chute, 1979). That the pattern of drug sensitivity in a population of coccidia can be greatly altered by the introduction of drug-sensitive parasites, and that this can be accomplished using some coccidiosis vaccines was first proposed by Jeffers (1976). The present results support this proposal.
In this study, vaccination was carried out for four flocks before changing back to a drug program and evaluating the sensitivity to drugs of any coccidia present. In the case of Coccivac-B®, restoration of drug sensitivity was demonstrated after two vaccinated flocks (Chapman and Jeffers, 2015; Mathis and Broussard, 2006). The optimal number of cycles of vaccination to achieve sensitivity restoration may vary depending upon the extend to which resistance is already present, the vaccine employed and local factors of epizootiological origin (Chapman et al., 2002). It is concluded that control programs, involving the alternation of chemotherapy and vaccination, may play a valuable role in the sustainable control of coccidiosis.
References are available on request
