The integrity of the gut is fundamental in birds as the mechanisms of digestion and absorption are inherent in the physiological metabolism of the gastrointestinal tract (GIT) and cannot be manipulated1. Numerous infectious and non-infectious agents can damage intestinal mucosa, as well as compromise digestion and absorption2. Among them, coccidia infections represent one of the most important enteric diseases. Coccidiosis is an infectious disease caused by a protozoan of the genus Eimeria (Apicomplexa: Eimeriidae) and is characterized by causing enteritis in various degrees in different segments of the intestinal tract, therefore decreasing performance parameters and increasing mortality3. Laboratory diagnosis of coccidiosis in poultry requires the identification of the species involved and quantification of the intensity of their infection by assessing the morphology, fecal oocyst count, location of the parasites in the gut, intensity of macroscopic lesions, prepatent period and oocyst sporulation time4. In the field, coccidiosis is diagnosed by assessing macroscopic lesions in the intestine of broilers. Eimeria acervulina, Eimeria maxima and Eimeria tenella are the species monitored on a regular basis5,6. A previous study showed that E. acervulina was the most prevalent (mean of 13.5%) species in Brazilian poultry operations, followed by E. maxima (6.75%) and E. tenella (4.35%)7. The purpose of the present study was to repeat and extend the evaluation and correlation of lesions caused by Eimeria with the other changes found in the gastrointestinal tract of broilers and the prevalence of coccidiosis in two production phases (phase 1: 12-21 days of age and phase 2: 22-40 days of age) in commercial poultry operations in Brazil between the years 2015 and 2016.
MATERIALS AND METHODS
Intestinal health monitoring was conducted in 72 broiler integration businesses in Brazil in the states of Rio Grande do Sul, Santa Catarina, Paraná, Mato Grosso do Sul, São Paulo, Minas Gerai, Rio de Janeiro, Goiás, Distrito Federal, Alagoas, Pará and Paraíba during the period 2015-2016. The data were posted in the Intestinal Health Program (PSI) of Vetanco of Brazil in order to obtain the percentage of affected birds and their ratings according to lesion scores. The analyzed data were divided into two stages of production: The 1st phase from 12-21 days old and the 2nd phase from 22-40 days old. Hence, this was an observational study. The chickens evaluated received diets prepared by their respective integrated companies without any interference of the appraiser in the formulation and use of performance enhancers and anticoccidial drugs used at the time of evaluation.
The most common anticoccidial program is the dual system, which uses a drug in the first phase (1st-21st day of age) and another one in the second phase (22nd day until product withdrawal limit). This program reduces the possibility of parasite resistance, a fact that extends the time that the drug were efficacious in the field8 and ensures more effective anticoccidial programs. To monitor the intestinal health of broilers, at least three birds/flock were evaluated. The birds were collected randomly inside the house at three different points (entry, middle and end).
In the intestinal analysis, the following lesions were observed: Presence of cell desquamation, excess fluid and mucus, ingestion of contaminated litter, thickening or thinning of the intestinal walls, Turkish towel appearance and necrotic enteritis. Injuries caused by Eimeria acervulina, Eimeria maxima and Eimeria tenella were scored according to their degree of intensity as specified by Johnson and Reids method,9 where the zero score indicates the absence of lesions and four indicates severe injury.
For microscopic assessments of E. maxima, the scraped intestinal mucosa technique for oocysts count was performed at the bowel portion next to Meckel's diverticulum. Costa and Paiva5 described that the highest concentration of E. maxima is found in the jejunum and Meckels diverticulum but can occur in the duodenum and at the end of the ileum. The content of the region was smeared on the microscope slide and a cover slip was placed over the content by pressing it gently. These slides were subjected to visual evaluation under the microscope at 100X magnification for oocyst counts at five different points (the four corners and center). The microscopic scores were ranked from 0-4, where 0 was the absence of oocysts, score 1 was 1-10 oocysts, score 2 was 11-20 oocysts, score 3 was 21-40 oocysts and score 4 was more than 41 oocysts10.
Data were analyzed using the Pearson correlation coefficient SAS11 with a 95% confidence interval.
According to the macroscopic analysis of lesions shown in Fig. 1, Eimeria acervulina was the most frequently observed species in both production phases. In phase 1, lesions related to E. acervulina were found in 8.8% of the necropsied broilers and were positively correlated (Table 1) with findings of cell desquamation, excess fluid, mucus, litter intake, thin intestine, feed passage, Turkish towel and duodenitis.
|Fig. 1:||Percentage of Eimeria in 1st and 2nd broiler production phases from 2015-2016
|Table 1:||Positive statistical correlations (p<0.05) of broilers affected by the factors that decrease intestinal health in relation to Eimeria in 1st and 2nd phase from 2015-2016*
|*1st phase (12-21 days of age), 2nd phase (22-40 days of age), **Microscopic assessment of E. maxima in the intestinal mucosa|
In phase 2, E. acervulina occurred in 18.2% of birds with its occurrence positively correlated with findings of cell desquamation, excess fluid, litter intake and feed passage.
E. maxima was the second most frequently observed species in affected birds. In phase 1, this coccidia was present in 4.3% of the birds. There was an increase in the prevalence of lesions caused by this species in phase 2, where it struck 6.9% of the birds. Lesions from E. maxima were positively correlated with cell desquamation, excess fluid, litter intake, feed passage, altered intestinal tonus, Turkish towel and E. acervulina.
E. tenella had the lowest prevalence in phase 1 and phase 2 (1.8 and 2.6%, respectively). There was a positive correlation between the occurrence of lesions caused by E. tenella in phase 1 with E. maxima and the occurrence of macroscopic lesions of E. acervulina in phase 2.
Under microscopic evaluation, E. maxima was present in 30.4% of the mucosal scrapings evaluated in phase 1, which represented a subclinical coccidiosis of 706.98% or 7.07 times in relation to clinical coccidiosis (macroscopic lesions of E. maxima according to Johnson and Reid).9
|Fig. 2:||Percentage of Eimeria scores in 1st and 2nd broiler production phases from 2015-2016
There was a positive correlation between the occurrence of E. maxima micro with findings of excess fluid, duodenitis and the occurrence of macroscopic lesions of E. acervulina and E. maxima. In phase 2, there was a significant increase in the occurrence of E. maxima micro, where 34.3% of the assessed mucosal scrapings showed oocysts of E. maxima. This prevalence represented a subclinical coccidiosis of 497.11% or 4.98 times compared to clinical coccidiosis. There was a positive correlation between the occurrence of E. maxima micro with cell desquamation, excess fluid, litter intake, feed passage, Turkish towel and macroscopic lesions of E. maxima. The data showed an increase in subclinical coccidiosis occurrences along the birds lifespan in that there was an increase of 112.84% or 1.13 times in the number of affected birds in phase 2 compared to phase 1.
Figure 2 demonstrates that level 1 of the macroscopic lesion score was the most commonly observed in both phases of production (1 and 2) for all evaluated species of Eimeria, while level 4, which is the most severe lesion score according to Johnson and Reid9 was present in a very small number of birds in the macroscopic evaluation. However, regarding micro scores for E. maxima, the score of level 4 was present in a significant number of birds (9.7 and 8.3% in phases 1 and 2, respectively). This represents a large number of oocysts present in the intestinal mucosa. Once eliminated in the bedding, these oocysts promote poultry recontamination and can lead to a higher chance of infection in other birds in the flock. Having large numbers of oocysts in the bedding leads to a greater chance of coccidiosis occurrence in its clinical form.
The prevalence of E. acervulina (13.5%) is in agreement with our previous findings7, which described this species as the most prevalent species of Eimeria in Brazilian poultry commercial operations. However, in a similar study, Do Amaral and Otutumi6 described an average prevalence of only 8.21% for E. acervulina.
Gazoni12 reported an average prevalence of 5.12% for E. maxima. This number increased to 6.75% by Gazoni et al.7, although these percentages are lower than the 8.52% reported by Do Amaral and Otutumi6.
The average occurrence of E. tenella reported by Gazoni12 was 11.47%, whereas Do Amaral and Otutumi6 claimed that E. tenella presented lesions in 2.42% of the birds. The result described by Gazoni12 for E. tenella is well above the values reported in this study (mean 2.2%) and by Do Amaral and Otutumi.6 These differences can be explained by the different general disturbances in the gut as disbacterioses, viruses and mycotoxicoses in each evaluation.
Due to a lack of maturity of their immune system, young birds are the most susceptible to clinical infections that often occur in asymptomatic adult birds, who spread the parasite. However, severe outbreaks can occur in adult birds as demonstrated in this study, in which there was a higher percentage of birds affected by the described species of Eimeria in the second phase. The route of infection is fecal-oral and the sporulated oocyst is the infective form. For the oocysts to sporulate, moderate temperatures (between 18 and 30̊C), relative humidity greater than 70% and well- oxygenated conditions are required. The average time of sporulation is approximately 17 h for E. acervulina, 18 h for E. tenella and 30 h for E. maxima4.
In contrast with our previous findings which showed that the micro scores of E. maxima had a prevalence of 23.8%7, the scores reported here had an average prevalence of 32.35%. This represents an increase of 135.71% in the last two years. According to Ito et al.2, intestinal scraping is the most suitable method for subclinical coccidiosis detection.
Teeter13 evaluated the impact of coccidiosis challenge on the poultry growth curve. In Teeter's work13, it was observed that for each point of increase in microscopic evaluation of coccidiosis lesions, daily body weight dropped 1.5% during the challenge period of 6 days. Therefore, it is important to know the degree of lesion to quantify the performance loss in the birds. It is estimated that during the coccidia asexual and sexual phases, each ingested oocyst destroys 2,048 enterocytes14. Hence, the economic importance of coccidiosis goes far beyond mortality and is mainly related to subclinical losses of asymptomatic animals, which maintain environmental contamination through oocyst elimination in their feces. Birds that did not exhibit clinical disease can also become uneconomical due to malabsorption syndrome1. Therefore, it is important to monitor the gut health of the birds by performing necropsies and mucosal scrapings to provide an early diagnosis of coccidiosis. Then, situations where anticoccidial programs are inefficient can be detected and preventive or therapeutic measures can be taken, providing coccidiocide drugs via drinking water14.
During the monitored period of two years (2015 and 2016), the macroscopic evaluation of lesions related to coccidiosis demonstrated that E. acervulina is still the most prevalent species in both phases of production analyzed (phase 1 from 12-21 days old and phase 2 from 22-40 days old), affecting on average 13.5% of broilers.
The occurrence of lesions caused by Eimeria positively correlated with several factors that decrease the intestinal health in two production stages, including cell desquamation, excess fluid, mucus, litter intake, thin intestine, feed passage, altered intestinal tonus, Turkish towel and duodenitis.
The lesion score level 1 was the most frequent score in both the 1st and the 2nd production phases. The subclinical coccidiosis, detected by mucosal scrapings microscopy, was observed in most of the chickens evaluated (mean 32.3%) and is one of the probable factors causing a reduction in the growth performance of broiler flocks in Brazil.
The analysis of necropsy findings and microscopy of the two evaluation phases demonstrated that the second phase (22-40 days of age) presented more severe lesion scores (scores 3 and 4). Hence, we have the greatest productive performance loss of broiler flocks in this stage.
It is vital that professionals in the poultry sector perform the intestinal tract evaluations mainly in the second phase (22-40 days of age) and if necessary, they may intervene in order to maintain the productive performance of broilers.
The authors would like to thank Vetanco of Brazil for funding this project and are also grateful to the Post-Graduate program in Veterinary Medicine of the Federal University of Santa Maria, Rio Grande do Sul - Brazil.