Periodicity of Ascaridia galli egg excretion in experimentally infected chicken in the Philippines

Abstract

The periodicity of parasite egg excretion refers to variations in the number of eggs produced across time, with significant implications in optimizing diagnostic procedures and conducting the Fecal Egg Count Reduction Test (FECRT). Here, we explore whether Ascaridia galli egg excretion varies across time under Philippine conditions, thus informing the best time to collect fecal samples during flock health examination. A time-course analysis was performed in chickens (N = 12) experimentally infected with A. galli, isolated from a naturally infected Philippine native chicken. We examined the fecal egg per gram (EPG) count at 3-h intervals for 3 days, starting from 5:00–6:00 h AM to the following day at 1:00–2:00 h AM. Our results showed a consistent daily egg excretion pattern with a peak EPG count in the morning that abruptly declined in the afternoon and lowest in the evening. The EPG counts correlated with the amount of excreta produced, suggesting that A. galli fecundity corresponds to the timing of host defecation. Our results imply that the best time to collect fecal samples for A. galli diagnosis and FECRT in Philippine conditions should be from sunrise until late morning when parasite EPG count and host excreta production are at their highest.

Introduction

In developing countries, backyard poultry production remains affected by the highly prevalent parasitic nematodes Ascaridia galli causing cachexia and intestinal impaction. The nematode is of significant concern in a free-range production system as it is effective in infecting new hosts due to its direct life cycle and environmentally adaptive infective stages (Sharma et al. 2019). In the Philippines, A. galli is one of the most prevalent nematodes in backyard chickens and in small-scale layer farms, causing increased mortality, reduced welfare, and low meat and egg production (Cormanes et al. 2016; Ybañez et al. 2018).

The diagnosis of A. galli in live birds relies mainly on fecalysis by demonstrating oval eggs with thick eggshells filled with granular blastomeres (Permin and Hansen 1998; Feyera et al. 2020). However, finding A. galli eggs in feces depends on the density of adult worms or the severity of the parasite infection. Moreover, the density of eggs varies depending on the parasite’s fecundity, host resistance, and the detection limit of egg counting techniques (Nielsen 2021; Morgan et al. 2022). This difference in the number of fecal parasite eggs across time is called the periodicity of nematode egg excretion, which is associated with spread and transmission in the environment (Daş et al. 2019).

Previous studies have demonstrated that temporal variations of parasite egg excretion may occur in some parasites under various conditions, such as in the nematode species A. galli (Oju and Mpoame 2006; Wongrak et al. 2015), Heterakis gallinarum (Wongrak et al. 2015; Daş et al. 2019), Aonchotheca caudinflata (Villanúa et al. 2006), and Syngamus trachea (Oju and Mpoame 2006), and also in the trematode species Schistosoma japonicum(Giver et al. 2000) and Echinostoma caproni (Platt et al. 2013). These studies imply that different parasites follow a rhythmic cycle on the release of their eggs depending on the host and the environmental or climatic factors, likely leaning toward an evolutionary adaptation for effective transmission (Platt et al. 2013; Daş et al. 2019).

Importantly, the periodicity of parasite egg excretion has a diagnostic implication that underlies timing for sample collection to maximize the chances of detecting parasite eggs, thereby decreasing the odds of a false negative result. This consideration is particularly significant when performing fecalysis using a method with a relatively low detection limit (e.g., 20–50 eggs per gram for the McMaster technique) (Vadlejch et al. 2011; Nielsen 2015; Zajac et al. 2021). To inform veterinarians on the proper timing for fecal sample collection during a flock health examination, our study explores the temporal variations of A. galli egg excretion under Philippines conditions and identifies the best time to collect fecal samples.

Materials and methods

Ascaridia galli egg culture

An 8-month-old naturally infected Philippine native chicken (nondescript indigenous type) was necropsied to collect the adult female A. galli from the small intestine. Ascaridia galli nematodes were identified based on their morphological features as described previously (Kates and Colglazier 1970; Zajac et al. 2021). The nematodes were macerated to collect A. galli eggs while immersed in 0.9% normal saline. The macerated nematode tissus were filtered using a wire mesh (500 μm), and the filtrate was examined for A. galli eggs. The A. galli eggs suspension was incubated at 28 °C for 21 days to achieve egg embryonation and monitored regularly for viability based on morphological appearance (Feyera et al. 2020). Cultured embryonated eggs were centrifuged at 100×g for 3 min, and the pellet was collected and washed with normal saline three times. The number of eggs for oral inoculation in chicken was counted using a Danish Bilharziasis Laboratory (DBL) slide (Carabin et al. 2005).

Experimental infection in chickens

One-day-old Starbro chickens (N = 12) were acclimatized for 3 weeks in a confinement area designed for nematode-free conditions. The individual chicken was confined in a 52 cm (length) x 36 cm (width) x 52 cm (height) cage with ad libitum access to water and feeds. They were fed a commercial diet containing 18–22% CP, 4–6% fat, 3–6% fiber, 0.8-1.0% calcium, 0.70% phosphorus, and 12% moisture. Individual water and feed troughs were refilled four times daily at 6:00, 12:00, 17:00, and 22:00. Experimental chickens received a natural light with a ~ 12 h day-night photoperiod, 27–28 °C environmental temperature, and 75–89% relative humidity, which are the natural climatic condition in the study site. After 3 weeks of acclimatization, individual broilers were experimentally infected with 50 embryonated A. galli eggs (infective stage) via the oral route.

Fecal egg count

After 4 weeks of experimental infection (i.e., observed prepatent period), fecal samples of each 10-week-old chicken were collected and examined daily for A. galli eggs using a flotation technique (Permin and Hansen 1998). After 31 days post-infection, the fecal egg per gram (EPG) count was monitored for 3 days at 4-h intervals: 5:00–6:00 h, 9:00–10:00 h, 13:00–14:00 h, 17:00–18:00 h, 21:00–22:00 h, and 1:00–2:00 h. To determine the EPG, two grams of fecal sample was collected and processed for flotation technique using a saturated sodium chloride solution (specific gravity of 1.2). The number of eggs per gram was counted using a modified McMaster egg counting technique, with a minimum detection limit of 50 EPG. Three technical replicates were prepared to derive the average EPG count per sample.

Data analysis

The EPG data was recorded and organized in Microsoft Excel. Log₂ transformation was used after performing the Shapiro–Wilk test, showing a deviation of data from the normal distribution (p < 0.05). After determining an equal variance using Levene’s test (p > 0.05), one-way ANOVA with Tukey HSD as a post hoc test was used to compare the average EPG count between time points. We also conducted a linear regression analysis to determine the association between excreta production in grams and EPG count. All analyses were performed in GraphPad Prism version 8 (CA, USA).

Results

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Temporal pattern of A. galli egg excretion

To determine the temporal pattern of A. galli egg excretion, we collected fecal samples at 3 h intervals for a three-day period, starting from 5:00–6:00 h AM to the following day at 1:00–2:00 h AM (Fig. 1A). Our fecalysis showed a significant peak of EPG count at 5:00–6:00 h in the morning that dramatically declined over time, with the lowest EPG at 21:00–22:00 h in the evening (Fig. 1B). With the highest EPG at 5:00–6:00 h AM as a comparator, we observed a fold-change of 1.21, 1.82, 4.65, and 13.21 upon reaching the time points 9:00–10:00 h, 13:00–14:00 h, 17:00–18:00 h, and 21:00–22:00 h, respectively, implying an increasing gap between EPG count from the morning to evening time points (Fig. 2A). Although the 9:00–10:00 h AM time point showed a relatively lower EPG count compared to 5:00–6:00 h AM, their EPG counts did not significantly differ (p = 0.399), suggesting that egg excretions from 5:00 h to 10:00 h AM pose relatively minor variations.

Fig. 1.

The daily pattern of A. galli egg excretion. A Flow diagram of experimental design indicating the time from acclimatization to the proper experiment. B The pattern of parasite egg per gram (EPG) across time points with repeated analysis for 3 days. The red dots represent individual EPG, while the blue dots represent the mean with SEM (blue shades)

Fig. 2.

Egg per gram (EPG) counts analysis. A EPG count comparison between time points. Purple dots represent the three-day average EPG count of the individual animal. The p values were calculated using one-way ANOVA with Tukey HSD post hoc test. B Linear regression analysis between excreta (grams) and EPG count showing the R² and p-value

We also observed that EPG counts started to rise at 1:00–2:00 h AM when referencing the previous evening time point at 21:00–22:00 h (p < 0.0001). This result suggests that parasite egg excretion may start early before sunrise and peak during sunrise (~ 5:00–6:00 AM) under Philippine conditions. However, the 1:00–2:00 h AM EPG count was two-fold lower than the 5:00–6:00 h AM, where the peak of EPG count was observed (p < 0.0001). We repeated the EPG counting at each similar time point for 3 days and observed a consistent trend (Fig. 1B), suggesting that A. galii egg excretion follows a rhythmic daily pattern. Overall, this result indicates that A. galli egg excretion under the Philippine setting follows a consistent diurnal cycle in which EPG counts peaked in the morning, abruptly declined in the afternoon, and significantly decreased in the evening.

Chicken excreta production and A. galli egg excretion

To determine whether A. galli eggs in feces correlate with chicken excreta (mixture of feces and urine), we performed a linear regression analysis between EPG count and grams of excreta produced per chicken at given time points (Fig. 2B). Our results showed a significant positive association between EPG counts and excreta production (R² = 0.5981; p < 0.0001). This indicates that lower excreta production was associated with lower EPG counts or higher excreta production was associated with higher EPG counts. Of note, in this study, EPG counts peaked in the morning (5:00–6:00 h AM) and dramatically decreased until late evening (21:00–22:00 h PM), thereby overlapping with a higher excreta production in the morning and declining excreta production in the afternoon and evening.

Discussion

This study investigates whether temporal variations of egg excretion in A. galli occur under Philippine conditions for fecal sampling optimization during flock health examination. Here, we observed that A. galli egg excretion under the Philippine setting follows a consistent diurnal cycle, with peak excretion in the morning, abruptly declining in the afternoon, and significantly decreasing in the evening. We also observed that the amount of A. galli eggs excreted correlates with the weight of excreta produced by chickens. The egg excretion pattern may likely be consistent daily in a patent A. galli infection under Philippine climatic conditions, as we observed its repeatability during repeated analysis for 3 days. Our results have two implications: (1) The timing of fecalysis when diagnosing parasites and conducting the Fecal Egg Count Reduction Test (FECRT) in chickens (Coles et al. 1992), and (2) providing support for the idea of rhythmic parasite egg excretion toward effective spreading and transmission (Kennedy 1976).

The FECRT is one of the methods used for assessing anthelmintic efficacy to evaluate a new synthetic drug or alternative medicine against parasitic helminths and for evaluating anthelmintic resistance of commonly used drugs (Fernandez et al. Jr 2013; Levecke et al. 2018). This method determines the reduction of parasite egg per gram (EPG) count over a specific time after treatment with a specific anthelmintic (Coles et al. 1992). Our results suggest that the timing of fecal sample collection is a significant factor that needs to be considered when performing FECRT in chickens. To avoid sampling bias for FECRT in A. galli under Philippine conditions, fecal samples should be collected consistently in the morning, when egg excretion and excreta are at the highest.

The peak egg excretion in our study has similarities in the western highlands of Cameroon in central Africa (Oju and Mpoame 2006), where the egg excretion pattern of A. galli in naturally infected native chickens peaked in the morning (8:00 h) and decreased at noon (12:00 h), possibly corresponding to the time when chickens are most active to feed and defecate. In contrast to our findings, Oju and Mpoame (2006) observed steady EPG counts in the afternoon (14:00–16:00 h) and another peak in the early evening (18:00) that correlates to a relatively low temperature and high humidity associated with afternoon rain. An early evening EPG peak in the western highlands of Cameroon may correspond to a favorable environment to disperse nematode eggs by avoiding desiccation (Oju and Mpoame 2006). Moreover, a study in Europe found that A. galli egg excretion from naturally and experimentally infected chickens was significantly highest in the late morning (10:00 h), with sustained EPG counts until the early evening (18:00 h), overlapping with the degree of chicken excreta production (Wongrak et al. 2015).

Apart from A. galli, the periodicity of egg excretion has also been reported in other parasitic nematodes affecting avian species. The chicken cecal nematode Heterakis gallinarum has been shown to excrete more eggs during the day than at night, of which EPG levels are synchronous with the degree of excreta production (Daş et al. 2019). The capillarid nematode Aonchoteca caudinflata in naturally infected red-legged partridge showed an increasing EPG count from morning to evening, with peak egg excretion at 19:30 h in the evening and lowest at 8:00 h in the morning (Villanúa et al. 2006). The chicken gapeworm Syngamus trachea in naturally infected chicken showed a peak of egg excretion in the late afternoon, while the lowest was in the morning (Oju and Mpoame 2006).

Overall, it is likely that the pattern of egg excretion in A. galli may differ between settings, possibly due to various factors, such as host and climate. For instance, parasite egg excretion correlates with the amount of excreta production, which is linked to the activity of the host (Platt et al. 2013). In other words, the fecundity of A. galli is likely in synch with chicken defecation timing (Wongrak et al. 2015), although defecation patterns may differ between geographical areas, possibly due to climatic factors. Climatic factors, such as temperature and season, greatly affect the host activity (Oju and Mpoame 2006) and likely, in turn, its defecation pattern. However, climate may have a minor effect in the Philippines with a tropical setting, in which slight variations in temperature with no distinct seasons are apparent. While no available published studies have been conducted in the Philippines that show seasonal trends for A. galli egg excretion, it is worth investigating whether rain and dry patterns have an influence on parasite egg shedding in the environment.

In the Philippines, A. galli is highly prevalent in scavenging backyard chickens and has been reported in small-scale layer farms in Leyte province, with an estimated prevalence of 41.2% (Ybañez et al. 2018). Since A. galli infection translates into poor production and loss of income, effective treatment and control measures will likely improve the production level of chicken farms by preventing economic losses associated with a low feed conversation ratio, poor body weight, and low egg production, as this parasite competes with chickens’ nutritional needs (Sharma et al. 2019). However, these treatment and control measures are anchored on proper diagnosis. Our results, therefore, inform the timing of properly diagnosing A. galli under Philippine conditions, especially when assessing the parasite burden of the flock, thereby optimizing the treatment and control strategy of the farm against this parasite.

Conclusion

In conclusion, A. galli egg excretion follows a consistent daily temporal pattern that correlates with excreta production and varies on the time of the day. Under Philippine conditions, our results imply that the best time to collect fecal samples for A. galli diagnosis and FECRT should be from sunrise until late morning.

Author contributions

HPP contributed to the study conceptualization and design, supervised the conduct of the study, performed data analyses, organized data figures, wrote the first draft, and contributed to the final writing of the manuscript. ILT collected the samples, performed laboratory analysis, and organized the data. TJF Jr conceptualized and designed the study, supervised the conduct of the study, and contributed to manuscript writing.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving animals

The CVM-VSU Research Committee has reviewed and approved the conduct of the study. The experiment was conducted following the Guide for the Care and Use of Agricultural Animals in Research and Teaching (FASS, 3rd Ed., 2010).