An Outbreak of Intestinal Obstruction by Ascaridia Galli in Broilers in Minas Gerais

Industrial broilers raised on helminthic medication-free feed were diagnosed with a severe disease caused by Ascaridia galli, characterized by intestinal hemorrhage and obstruction. A. galli was identified based on the morphological features of the nematode. Broilers were raised for a longer period (63 days) for weight recovery, grouped as stunted (n=500), had low body score and had fetid diarrhea. The duodenumjejunum segment was the most severely affected with obstruction and had localized accumulation of gas. The intestinal mucosa was severely congested with petechial and suffusive hemorrhages. The outbreak resulted in morbidity of about 10% and mortality of up to 4% and was associated to the absence of preventive medication on feed and slack biosecurity. The reemergence of A. galli is discussed in view of the alternative poultry management and raising conditions for drug free and welfare.


INTRODUCTION
Ascaridia galli is returning to the relevant poultry diseases scene as alternative free-range broilers and cage-free layer chickens are progressively in higher demand by consumers. Floor and ground conditions enable the direct ingestion of eggs or larvae or mechanical vectors of Ascaridia galli, such as earthworms, which may concentrate and protect eggs (Augustine, 1974), although the transmission is mainly direct and the life cycle simple. Eggs develop infective larvae within about 12 days and are resistant to cold. The normal development and viability of A. galli eggs is highest in anaerobic conditions in feces at cold temperatures but lowest in feces or water at warmer temperatures, as eggs maintained in normal embryogenesis with little loss of viability in feces at 4°C, but with large viability losses in clean water at 25°C (Tarbiat et al., 2018). Infection will result in reduced productive performance of meat or eggs, and high parasitic load will cause intestinal blood loss, increased mortality, and anemia, and may aggravate other conditions (Norton & Ruff, 2003).
In Germany, cage-free organic layers were shown to harbor, mostly subclinically, the nematodes Ascaridia galli (88%), Heterakis gallinarum (98%), and Capillaria spp (75.3%) (Kaufmann et al., 2011). In Sweden, layers are challenged with genetically related Ascaridia galli strains, indicating recent and stationary flock infections (Höglund et al., 2012). A more extensive study in eight European countries investigated helminthic infection, revealing A. galli with an overall prevalence of 69.5%, and that, in contrast to the general assumption, the outdoor pasture did not correlate to the infection (Thapa et al., 2015).
In Brazil (São Paulo), adult chickens of different housing and management strategies, were shown most commonly infected by A. galli and Heterakis gallinarum (Silva et al., 2016). An outbreak of intestinal disease in Pavo cristatus causing diarrhea, dehydration, anorexia and weight loss, was associated to A. galli (Teixeira et al., 2012). Wild native and exotic domestic avian species were investigated for the presence of endoparasites in feces and found A. galli in Buteo magnirostris and P. cristatus (Marietto-Gonçalves et al., 2009;Andery et al., 2013).
This manuscript aims to report an outbreak of severe infection by Ascaridia galli resulting in intestinal hemorrhage and obstruction and high mortality of industrial broilers raised on medication-free feed.

MATERIALS AND METHODS
Broilers (n=4) which were raised up to nine weeks of age were received for diagnosis, with history of morbidity of approximately 10% and mortality of approximately 4%. The flock was formed by gathering stunted chicks for separate management and characterized by low performance, stunted growth and low body weight. All birds in the sample were chosen considering the stunted growth. Broilers were raised on balanced nutrient but helminthic medication-free diet, with a total of 500 birds in the flock. Housing was in conventional brick house for industrial broilers, with industrial grade feeders and drinkers, forced ventilation, curtains, cemented floor and wood shavings bedding. However, biosecurity was not effective, without entrance control, allowing visitation, with no footbath or vehicle disinfection. Complete necropsies were performed, and nematodes collected, processed, mounted as described previously (Bowman, 2009), and identified according to the morphology and micrometry (Soulsby, 1982). Briefly, nematodes were immersed in warm buffered saline (PBS pH 7.4) and within a few minutes were subsequently transferred to a buffered formaline solution (formaldehyde 10% in PBS). Helminths were diaphanized in lactophenol (20% phenic acid, 20% lactic acid, 40% glycerine; 20% distilled water) and mounted onto glass slides with coverslip in melt phenol gelatin (10g gelatin; 60mL distillled water; 70ml glycerine; 0.5mL phenic acid) and visualized with an optical microscopy (Olympus B-H2) at 40 and 100 magnifications.

RESULTS AND DISCUSSION
Broilers were described with chronic fluid fetid diarrhea and showed low body score for the age, with prominent carina (sternum), as a result of reduced pectoral mass. Enlarged intestines with gas accumulations were seen at necropsy, where Ascaridia galli concentrated, mostly at the cranial part of the jejunum, causing obstruction and mucosal congestion (Fig. 1). The microscopic aspects of the morphology of A. galli adults and eggs are shown (Fig. 2).

eRBCA-2019-1072
Ascaridia infections are known to be rare in caged chickens, including welfare cages, but common in non-caged systems, as here described for indoor broilers. In the outbreak here described, broilers were raised in conventional houses, but without adequate biosecurity, such as entrance control for visitors or vehicle disinfection, and were given feed with no anthelminthic medication. The improvement of management factors such as housing, litter and nutritional quality, schedule times of opening and closing popholes, alternation of pasture and supply of required materials may have a role in the reduction of the helminthic load (Thapa et al., 2015), as freerange conditions enable the ingestion of eggs or larvae, or mechanical vectors of Ascaridia galli, such as earthworms (Augustine, 1974). Although free-range on grass did not correlate to Ascaridia galli infection in Europe (Thapa et al., 2015), despite temperatures of 18.8°C or lower will stop larva development (Reid, 1960), but preserving its viability (Tharbiat et al., 2018), the climatic and environmental conditions may have a role in other regions, such as cooler climates. A. galli has a broad avian host range and infection has been described in industrial and free-range chickens, and wild species (Marietto-Gonçalves et al., 2009;Silva et al., 2016;Teixeira et al., 2012;Andery et al., 2013).
Lesions by A. galli will harm intestinal function and performance, with retarded growth and production (Daş et al., 2012), especially by larval stages (Luna-Olivares et al., 2015), as here observed as intestinal hemorrhages and obstruction.
The introduction of Ascaridia galli infection in the premises might be associated to infected replacement chickens (Höglund et al., 2011), although the established infection will maintain a local genetic profile identity, and with genetic variations among farms (Höglund et al., 2012). Broilers in the present outbreak were raised in premises without entrance control, hence insufficient biosecurity. Visitor entry to the house or unit is a management error which results in eggs introduced horizontally into the farms (Jansson et al., 2010).
Ascaridiosis seems to be reemerging in several countries due to alternative raising or housing. In Serbia, hens of organic egg production were found 15.6-24.0% positive for A. galli (Tamara et al., 2019). In Argentina, broiler chicken flocks were evaluated and 25% of flocks were shown infected but no correlation was found between nematodes and coccidian infections (De Franceschi et al., 2008). Previous studies in Brazil, have found broiler flocks infected by Ascaridia galli in 10/17 municipalities of São Paulo (Da Silva et al., 2018), in free-range flocks in the neighboring state (Paraná), with 45% infected (Vieira et al., 2015), and in Santa Catarina in 21.7% (Quadros et al., 2015). In our study, we show that raising broilers in Minas Gerais, previously shown to naturally occur A galli by Mendes et al. (1976) and in our unpublished extension service, especially for longer and without preventive medication, may lead to highly significant infection.
The information available on the affected flock revealed the strategic use of mebendazole for treatment against ascaridiasis. However, more effective drugs have been recommended against A. galli, such as fenbendazole, dl-tetramizole, levamizole or pyrantel tartrate (Norton & Ruff, 2003), none of which are permitted in drug-free poultry.
The climatic conditions and change in Brazil may favor parasitism in most of the Brazilian territory, with the survival of eggs in feces, especially at cooler temperatures (Tharbiat et al., 2018). The adoption of medication-free diet will demand for frequent fecal monitoring and strict biosecurity, in order to reduce the risk of helminthiasis. However, the use of antihelmintics has been considered a selective pressure for resistance. For instance, the subtherapeutic preventive dosage with benzimidazole has been involved in resistance by Ostertagia ostertagi in bovines (Knapp-Lawitzke et al. 2015).

CONCLUSIONS
The parasitism by Ascaridia galli in broilers is a growing concern worldwide, especially for alternative poultry, or in drug-free raising of broilers, as shown here. The growth of industrial broilers in medicationfree diets will demand for all the adequate preventive veterinary medicine measures to be taken.