INTRODUCTIONThe production of meat-type poultry has greatly expanded over the past several decades. The commercial broiler industry has evolved from backyard flocks into an ingenious mass food production system. Growth and yield of birds have been enhanced with the help of highly specialized diets and through the use of genetics. At the breeder level, superior birds are selected based on attributes desirable to the customer, while enhancing growth, reproductive rates, and an improved immune system of the bird. This ability to adapt the bird based on consumer preference while improving bird livability has allowed the industry to thrive and succeed.The primary focus of the commercial broiler industry is to maximize profits by promoting maximal yield and maintaining the health of the bird. Any hindrance to bird health will decrease profitability. Improvements in technology relating to vaccines or nutrition could save companies money and allow the industry to operate more efficiently by increasing revenue and decreasing overall costs. One of the main expenses faced by the industry is loss associated with poultry diseases, including costs of vaccination, prevention, treatment, reduction in weight gains, and mortality. Coccidiosis is a parasitic disease that is responsible for losses in production of food-producing animals worldwide. The incidence of coccidiosis in commercial poultry has increased due to higher stocking densities and more intensive husbandry practices. Stocking densities such as 0.7 ft2per bird, among other stressors, have favored the spread of this disease in commercial poultry facilities. It has been documented that coccidiosis is the most consistently reported health problem in poultry (Biggs, 1982; Rose et al., 1987; Williams, 1999).Over the past 100 years, much research has persisted on coccidiosis because of its significance in the animal industry. Researchers interested in the Eimeria species continue to analyze the intricate details of its life cycle in hopes of alleviating the potential hazards and economic losses caused by this disease. The current methods for control of coccidiosis can be attributed to knowledge gained through past research and investigations. Much of the host-parasite interaction is unclear; however, ongoing and future information obtained will help with the treatment, prevention, and perhaps the eradication of this costly disease.
--------------------------------------------------------------------------------
Page 2
2Our laboratory is particularly interested in the mechanisms responsible for mucosal immunity to coccidial parasites in broilers. Understanding these mechanisms has been complicated with the immunovariability associated with different isolates within the same Eimeria species. The purpose of the present study was to examine two different isolates of Eimeria acervulina and the pathology and immunovariability associated with both. Eimeria acervulina was chosen for the series of experiments because of its prevalence and continual occurrence in the commercial poultry industry. We hypothesized that immunovariability does exist between different strains within the same species of Eimeria and that there is a differential host response associated with each. In addition, mast cell numbers are increased during an Eimeria infection and exhibit effector functions, which mediate inflammatory responses and aid in intestinal immunity.
--------------------------------------------------------------------------------
Page 3
3REVIEW OF LITERATUREEimeriaCoccidiosis is a self-limiting, infectious disease of the digestive tract caused by host-specific intracellular protozoal parasites of the genus Eimeria. Coccidia are classified under the subkingdom Protozoa of the phylum Apicomplexia (Jeurissen et al., 1996; Lillehoj, 1996). As a group, coccidia of the genus Eimeria cause the most widespread health problems in the broiler industry and remain one of the most expensive diseases of commercial poultry production (Edgar, 1992; Henken et al.,1994; Yun et al.,2000). Birds infected with coccidial oocysts do not perform as well as non-infected birds as a result of moderate to severe damage to the intestinal mucosa. Birds exhibit decreased weight gains, increased feed conversion, and in some cases, birds may appear asymptomatic, but are limited in their ability to maximize feed efficiency. According to Edgar (1992), it takes only one viable oocyst to establish the presence of coccidia in a poultry house. This is possible because of the parasites reproduction index. Ingestion of one Eimeria acervulina (EA) oocyst in the infective state has the ability to yield about 72,000 oocysts in one complete life cycle (Henken, 1994). The prevalence of coccidiosis is worldwide and can be found in almost every commercial poultry flock (McDougald and Reid, 1997; Cox, 1998). In poultry, there are seven species of Eimeria that infect chickens (Shirley, 1986; McDougald and Reid, 1997). All of the chicken coccidia are pathogenic; however, some species produce more severe effects than others, such as severe morbidity and mortality. These microscopic, one-celled parasites invade their host via the fecal-oral route, and immunity is achieved once the parasite completes its life cycle in the host (Brackett and Bliznick, 1950; McDougald and Reid, 1997). Chickens of all ages are susceptible to coccidiosis, but birds that are three to five weeks of age are the most vulnerable (Edgar, 1992; Yun 2000). The Eimeria species have an extremely complex life cycle comprising stages both internal and external to the host. Infection occurs by ingesting sporulated coccidial oocysts found in contaminated litter, soil, feed or water. After ingestion, the protozoa go through a series of intracellular, extracellular, asexual, and sexual stages to produce viable oocysts that are excreted in the feces (Rose, 1987). After a brief period outside of the host, the oocysts become infective again through the process of sporulation, and the life cycle is complete.
--------------------------------------------------------------------------------
Page 4
4The impacts of coccidiosis are significant; however, eradication is impractical due to protective mechanisms of the oocysts. Eimeria possess a thick outer wall that acts as a protective barrier, which enhances the chance of survival under severe conditions. The oocysts are able to remain infective outside of the host for long periods, and their protective properties allow them to be resistant to many harsh chemicals and disinfectants (Eschenbacher et al., 1996; Jeurissen, 1996; Yun et al., 2000).Coccidian Life CycleAs the Eimeria species tend to be very specific in the intestinal region at which they invade, their life cycles are similar with a degree of species specificity. The Eimeriacomplete their life cycle in three distinctive phases including sporogony, merogony, (schizogony) and gametogony; however, the lengths of these phases are unique to the species (Yun et al., 2000). The following life cycle is adapted from Edgar (1992). Coccidiosis, caused by the ingestion of microscopic oocysts, is easily transmissible by mechanical means such as contaminated footwear and equipment, or it can be found in litter, contaminated soil, feed, or water (Conaway and McKenzie, 1991; McDougald and Reid, 1997). Chickens canbecome infected by coccidia once the oocysts develop into an infective stage outside of the host. Sporogony is the process of a one-celled zygote within the oocyst undergoing a series of divisions to form sporozoites, which are contained within sporocysts. Only oocysts that have undergone this process are able to cause disease. Sporulated oocysts contain four sporocysts, and each sporocyst contains two sporozoites. Mechanical action of the gizzard and pancreatic enzymes such as trypsin and bile salts cause the destruction of the oocysts outer wall, which releases the sporocysts into the digestive tract. The sporocysts are then further excysed by trypsin and bile salts that are present in the intestine. The sporozoites invade villus epithelial cells along specific locations throughout the digestive tract depending on the species of Eimeria. Some species travel within the mucosa, through the lamina propria to the crypt epithelial cells. Once inside villus or crypt cells, the process of merogony takes place. The sporozoite develops into a rounded body called a trophozoite, and then into an asexually reproductive first-generation schizont (meront). The schizont grows and divides rapidly to produce many first-generation merozoites. The bodies rupture and release hundreds of first-generation merozoites, which
--------------------------------------------------------------------------------
Page 5
5seek out and invade other epithelial cells. Second-generation trophozoites develop into second-generation schizonts. When the second-generation schizonts mature and rupture, increased quantities of invasive merozoites are released causing widespread infection. The number of asexual stages and time required for each depends on the Eimeria species involved; however, most species will have less than four asexual reproductive generations (Rose, 1987; Edgar, 1992). Merozoites produced by the latter schizont generations develop into sexual forms called gametocytes, some male and some female. This phase of sexual reproduction is termed gametogony. The female gametocyte matures into a macrogamete and the male gametocyte matures and ruptures releasing a large number of motile, biflagellate microgametes. The microgametes penetrate the mature female macrogamete and fertilization occurs. Following fertilization, a thickened protective wall forms around the zygote. At this stage, the zygote is considered an immature oocyst. When mature, the oocysts rupture the host cell, enter the lumen, and are expelled into the feces. Clinical signs are associated with tissue destruction from the release of the merozoites and mature oocysts from the mucosal surface during the last generations of merogony and throughout gametogony. In severe infections, much of the mucosal epithelium is sloughed off and nutrient absorption is compromised (Jeurissen, 1996; McDougald and Reid, 1997; Yun, 2000). Oocysts excreted from the birds remain in the environment and have the potential to infect other birds. Under favorable environmental conditions (approximately 84°F), sporulation of oocysts will be achieved in 24 to 48 hours, and the cycle will continue (Edgar, 1992; Graat et al., 1994; McDougald and Reid, 1997). According to Edgar (1992), once the oocyst is sporulated, it can remain infective to birds from several months to one or two years if protected from very hot, dry, or freezing conditions. Coccidial oocysts have rigid characteristics but are not totally indestructible. According to Lee (1988), unsporulated oocysts are more susceptible to physical and chemical agents than sporulated oocysts perhaps due to a highly sensitive metabolic state. Other factors, such as ammonia and anaerobic conditions, are also lethal to oocysts in the environment. Cessation of development occurs when oocysts are introduced to high levels of CO2or NH4,or exposed to mercury salts, mercuric cyanide, and mercuric chloride due to the ability of these agents to penetrate the oocyst wall (Kheysin, 1972). The prepatent period, the time it takes for oocysts to be seen in
--------------------------------------------------------------------------------
Page 6
6the feces after ingestion, is approximately 4 to 7 days for the Eimeria species (Henken et al., 1994; Jeurissen, 1996; McDougald and Reid, 1997). Coccidiosis in Commercial Broiler Operations Coccidiosis has plagued the poultry industry since the early 1900s, and by the 1940s, considerable information was gathered on the pathophysiology, immunology, epidemiology, and therapy of this disease. Large-scale production of poultry was developed, and with it came a need to study the control of threatening diseases (Brackett and Bliznick, 1950). Today, much knowledge has been gained about coccidiosis, but it continues to have a major economic impact on the commercial poultry industry. It has been reported that the US poultry industry suffers in excess of one to two billion dollars in annual losses relating to coccidial infection, treatment, and prevention (Danforth and Augustine, 1989; Talebi and Mulcahy, 1995; Yun et al., 2000). However, it is difficult to accurately estimate the total monetary losses suffered by the worlds poultry industry resulting from coccidiosis and its prevention or control (Danforth, 1998; Williams, 1999), because coccidiosis infects any type of poultry in any type of facility and its occurrence is worldwide (McDougald and Reid, 1997). Chicken coccidial species are highly species specific, and acquired immunity can be achieved once the coccidia complete their life cycle. However, birds can harbor the disease and be carriers after infection, increasing the likelihood of spreading coccidiosis (Lee and Shih, 1988; Williams, 1998). Most infections are mild due to the ingestion of few oocysts, and the disease will go unnoticed. With such infections, maximal feed efficiency and feed utilization by the bird is only slightly decreased. Ingestion of millions of oocysts, on the other hand, may cause severe infections, and a disastrous outbreak could occur. Flocks infected as a result of mild to severe exposure usually show a marked decrease in food and water consumption, and birds become depressed and tend to huddle. Decreased weight gains occur as a result of the disruption of the intestinal mucosa where minimal absorption is taking place. Diarrhea may result as the host is trying to flush the organism from the body, which may induce dehydration. Lesions of the intestinal mucosa and loss of pigmentation may also become apparent during the latter stages of infection (Conaway and McKenzie, 1991; Edgar, 1992; Lillehoj and Trout, 1993; McDougald and Reid, 1997).
--------------------------------------------------------------------------------
Page 7
7Mortality could result due to the lack of adequate nutrient intake, secondary infections, and other continual stressors associated with the diseased state. However, with the use of control agents such as medications, vaccines, or coccidiostats, the effects of coccidiosis are suppressed or even prevented. Since the 1950s, the control of coccidiosis has been achieved through anticoccidial compounds administered in the feed, which prevent or reduce infections to a sub-clinical level (Danforth, 1998). When used correctly, these compounds provide sufficient disease control. However, current management practices, such as intensive confinement, encourage the severity and transmission of coccidiosis. With the use of continual feed application of anticoccidials, unavoidable drug resistance by avian coccidia has resulted. This has allowed for an increased selection of drug-resistant strains, which reduce the efficacy of many anticoccidials that are used today. To further control the emergence of drug-resistant strains, shuttle and rotation programs have been pursued. These strategies have given good results in the past but continued efficacy is questionable. To date, the most serious limitation to anticoccidial therapy is the parasites increasing tolerance (McDougald and Reid, 1997). New and innovative anticoccidial products are unlikely to be marketed due to the increased costs of approving such compounds for food animals and consumer perspectives concerning feed additives (Danforth, 1998; Vermeulen, 2001). With such constraints, producers and researchers have tried to identify drug-free alternatives to coccidiosis control such as improved sanitation upon complete litter cleanout, selective breeding for improved immunity, and vaccination programs (Williams, 1998). However, developments of new and improved vaccines in recent years seem to have the greatest potential. In order for a commercial vaccine to be successful, it must have a reasonable cost, be as effective as other methods, give solid protection within a short period of time, and offer long-lasting immunity (Danforth and Augustine, 1989). To date, there are four vaccines available for commercial chicken flocks but their use in broilers is relatively low due to the short grow out period (Williams, 1998). The main objective of the broiler industry is to produce a high quality product at relatively low costs. Any extemporaneous costs related to treatment or prevention of enteric diseases increase production costs and lower profits. The drastic impacts on production caused by coccidiosis have significant economic effects at each facet of the industry. The
--------------------------------------------------------------------------------
Page 8
8goal of researchers and scientists is to sequester the effects of this enteric disease, which will in turn lower the overall costs of coccidiosis. Understanding the biological aspects of the host response to Eimeria infections at a cellular level is crucial to the development of new approaches to coccidia control.Eimeria acervulina In the poultry industry, nine species of the genus Eimeria have been described in chickens; however, in recent years, only seven of these species are known to exist and cause pathogenic effects (Conway and McKenzie 1991; Williams, 1998). Coccidial species are classified based on attributes such as: 1) intestinal location of infection, 2) appearance of intestinal lesions, 3) oocyst size, shape, and color, 4) average prepatent period length, 5) size of invasive sporozoites and merozoites, and 6) type of cell parasitized and location within epithelial cells. Based on these characteristics, E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox, and E. tenella are considered pathogenic for chickens. Of the seven species, E. acervulina (EA) has been one of the most frequently encountered coccidial parasites infecting poultry in commercial operations (Henken et al., 1994b; McDougald and Reid, 1997). Henken et al. (1994a) assumes that every flock is infected with coccidial parasites, and EA is responsible for the majority of these cases. Within the commercial broiler industry, infection with EA has major impacts on production. These impacts are related to how the host reacts to the parasite infection. However, there are other factors affecting the severity of disease such as the amount of viable EA oocysts ingested and pathogenicity of the isolate as well as the genetic, nutritional, and immune state of the bird (McDougald and Reid, 1997). A mild infection with EA could produce sub-clinical effects such as reduced feed utilization and increased feed passage, which can increase feeding costs and overall feed conversion ratios. However, a moderate to severe infection with EA can produce clinical signs such as loss of appetite, decreased water consumption, reduced weight gains, loss in skin pigmentation, diarrhea, lethargy, poor feed conversion, and whitish-gray lesions of the intestinal mucosa. Heavy infections may cause lesions to coalesce and death may occur (Johnson and Reid, 1970; Edgar, 1992; McDougald and Reid, 1997). While each species of Eimeria tend to be site specific, EA invades the anterior portion of the small intestine, specifically the duodenal loop and in severe infections, the anterior portion of the
--------------------------------------------------------------------------------
Page 9
9jejunum. It is speculated that secreted molecules from the invasion site itself and surface molecules located on the parasite could be responsible for this site specificity (Lillehoj and Trout 1993; Jeurissen et al., 1996).Immunity to Coccidial ParasitesAs described above, Eimeria reside outside the host for part of their life cycle but, the majority of it is completed inside the host during asexual and sexual stages of development occurring inside or outside enteric tissues. Once the bird ingests the viable oocyst(s), a cascade of events occur involving both non-specific and specific defense mechanisms of immunity (Lillehoj and Lillehoj, 2000). It is to be expected that the mechanisms responsible for immunity are complex due to the complexity of the parasite life cycle. Despite all of the research completed on immunity to Eimeria, no clear picture has emerged as to how complete resistance is acquired and which mechanisms are sequentially involved in generation of immunity (Rose et al.,1979; Danforth and Augustine, 1989). In naïve chickens, those previously unexposed to Eimeria, coccidial infections induce a variety of pathological and immunological responses, which help the host defend against the parasite and acquire protective immunity. However, the level of protection each facet of the immune system provides may vary with the developmental stage of the parasite (Rose, 1987). Prior to the generation of a specific immune response, the host tries to exclude the Eimeria through non-specific immune pathways such as competitive exclusion by normal flora, lysozymes, increased gastric secretions, and peristalsis to quickly flush parasites from the digestive tract (Lillehoj and Lillehoj, 2000; Yun et al., 2000). However, it has been reported that these innate defenses as well as specific immunologically mediated defenses play a role at the intestinal mucosal surface during Eimeria invasion (Lillehoj and Trout, 1993). Therefore, the naïve host probably does not eliminate the parasite utilizing only non-specific pathways, but infection can be controlled to a certain degree prior to the completion of the Eimeria life cycle and generation of a specific immune response.It has been determined that Eimeria parasites are vulnerable to the host immune system at three distinct phases of their development: 1) the period between excystation and sporozoite penetration of epithelium, 2) once the sporozoite enters the host epithelium and is exposed to intra-epithelial lymphocytes, and 3) during transport of the sporozoite from the
--------------------------------------------------------------------------------
Page 10
10surface enterocyte, through the lamina propria and into the crypt epithelium. After these stages in the life cycle, direct interaction between the parasite and cells of the host immune response are unlikely, and the probability of intervention is ceased (Jeurissen et al., 1996). During phases of the Eimeria life cycle that allow close contact with the immune system, both humoral and cell-mediated responses are stimulated; however, the contributions of each to protective immunity are still debated. The immune response to coccidial parasites has been extensively reviewed (Lillehoj and Trout, 1996; Yun et al., 2000). As discussed in these reviews and in other literature, the humoral arm of the immune system has been shown to play a role in conferring immunity to coccidial parasites, possibly more in the control of primary infections, as evidenced by studies with bursectomized and bursal-diseased chickens. It was shown that B-cell depleted animals are less resistant to primary infections and more susceptible to challenge than controls (Rose and Hesketh, 1979; Rose, 1987; Lillehoj and Trout, 1996). It has been suggested that species-specific circulating antibodies released into the lumen of infected birds have a protective effect by directly blocking invasion at the mucosal surface or by enhancing intraluminal destruction of sporozoites (Lillehoj and Trout, 1996). Serum from immune birds has also been shown to have a protective effect against coccidiosis when given to naïve birds (Rose, 1984; McDougald, 1985; Rose, 1987; Lillehoj and Trout, 1996; Yun et al., 2000). Although studies have shown that antibodies prevent initial invasion, it is less certain if they limit the course of disease once infection is established (Lillehoj, 1991). However, it is believed that cell-mediated immunity has a far greater role in protection against coccidial parasites than humoral immunity, but both systems are important for birds to acquire complete protective immunity (Rose, 1987; Jeurissen et al., 1996). Cell-mediated responses are an integral part of protective immunity as shown in experiments where T-cell depleted animals were unable to resist primary or secondary challenge infections (Rose and Hesketh, 1979; Rose, 1982a; Rose, 1987). Similar results were found when cyclosporin A (Cs-A), betamethasone, and dexamethasone, cell-mediated immune response suppressing agents, were given to naïve birds prior to Eimeria challenge (Lillehoj and Trout, 1993; Lillehoj and Trout, 1996). Talebi and Mulcahy (1995), observed a significant negative correlation between cellular lymphocyte responses and fecal shedding of oocysts in birds infected with homologous coccidia challenge, which supported the major
--------------------------------------------------------------------------------
Page 11
11role of cell-mediated immunity. Further studies have confirmed that T-cell sub-population numbers in the intestinal epithelium and lamina propria change during primary and secondary EA challenge, specifically, CD4+ T-cell increases during primary challenge and CD8+ T-cell increases during secondary exposure (Lillehoj and Trout, 1996). These results suggest that variations in T-cell sub-populations may reflect infection-related changes in the intestine. Martin et al. (1995) separated several fractions from sporozoites and merozoites in order to identify antigens from EA and how they contribute to protective immunity. It was determined that the synonymous effects of cell-mediated and humoral responses confer a species-specific protection, which lasts throughout the life of the bird. Protective immunity has normally been measured by maintenance of body weight gains, reduction in lesion scores, cessation in total oocyst output, and antibody and cellular responses in broiler birds (Stiff and Bafundo, 1993; Talebi and Mulcahy, 1995). While birds that have acquired protective immunity to a particular species of Eimeria no longer exhibit clinical signs of disease, they may continue to shed oocysts in the feces (Lee and Shih, 1988; Williams, 1998), a condition known as coccidiasis. However, Stiff and Bafundo (1993) showed conflicting results in a series of experiments that proved complete immunity can and does exist when birds are continuously challenged on a daily basis with homologous Eimeriachallenge. Nevertheless, protective immunity hinders fecal oocyst production, and invasion of the mucosal epithelium is altered. Interestingly, in immune birds, sporozoites penetrate the villus epithelium but are incapable of reaching the crypt epithelium and prevented from further development (Rose et al., 1984; Jeurissen et al., 1996).Physiological ChangesA closer look at histological and physiological changes that occur during the course of the immune response to coccidial parasites is pertinent to understand the specifics of immunity. Responses to Eimeria parasites at the intestinal mucosa may be mediated directly by the parasite or by the hosts immunoinflammatory response, which may result in changes in the intestinal mucosal morphology (Barker, 1993). Physiological changes related to infection have been well documented in experiments with EA infected chickens. Results indicate that at the level of the intestinal mucosa, local and systemic responses to coccidia are mediated in different ways. Fernando and McCraw (1973) measured the intestinal response
--------------------------------------------------------------------------------
Page 12
12to a single dose of EA, and infected birds showed a marked increase in total mucosal thickness, decreased villus height, and increased crypt length in the duodenum and to a lesser extent in the anterior jejunum. The mucosal alterations were the most severe at the height of infection, and a marked increase in the rate of replacement of intestinal epithelial cells was observed. In addition to an increase in duodenal epithelial cell turnover, Allen (1983) noticed an increase in metabolism of mucosal cells in the lower intestine, which may enhance compensatory growth and help the bird overcome the negative affects of infection on parameters of production. Other morphological changes were evident, such as increased gut length and increased tissue moisture (edema) in the intestine, which were not associated with starvation, but the sole effects of challenge. Changes in gut environment are also evident from challenge, such as decreased pH of the infected area (Ruff, 1984) and increased feed passage time (Stephens et al., 1974; McKenzie et al., 1986). These changes in gut homeostasis may alter feed intake, which leads to decreased nutrient digestion and absorption, changes in metabolism, and overall decreases in weight gains (Adams et al.,1996). Interestingly, researchers have contrasting results in experiments where weight gains are measured. Stephens et al. (1974) and Adams et al. (1996) reported decreased weight gains associated with EA challenge, while Ogbuokiri and Edgar (1985) and McKenzie et al. (1986) showed no significant weight gain differences when compared to non-challenged controls. This difference in results could be associated with several factors such as genetics of the birds, route of challenge administration, amount of infective oocysts administered for challenge, and strain of EA (Danforth and Augustine, 1989). Additional studies have shown that initial contact of the sporozoites with the intestinal mucosa produces an inflammatory response including marked cellular infiltration at the site of infection (Rose et al., 1979). This infiltrate consists of multiple leukocyte sub-populations, macrophages, natural killer cells, granulocytes, and lymphocytes that could modulate and enhance immune responses; therefore, altering nutrient absorption and decreasing weight gains (Jeurissen et al., 1996).The specific mechanisms by which each coccidial species causes disease is not clear; however, the establishment of secondary infections due to the altered intestinal mucosa may be responsible for a differential host response (Barker, 1993). This is possible because coccidia are able to interact with other pathogens such as bacteria and viruses, which may
--------------------------------------------------------------------------------
Page 13
13amplify the observed effects (Ruff, 1993). As stated above, the ability of the chicken to control the severity of infection and develop protective immunity to coccidial parasites depends on numerous factors. Host responses to the parasite involve a complex series of internal factors that are dependent on developmental stage of the parasite, immune status of the bird, and species and strain of the Eimeria parasite.Mast Cell Role in Coccidial ImmunityCurrent research has focused on alternative explanations for the mechanisms responsible for protective immunity to coccidial parasites. It is well established that many cells are involved in the response to Eimeria invasion, including rapid infiltration of polymorphonuclear cells (PMN), lymphocytes, and large mononuclear cells (LMN) to the site of inflammation (Rose et al., 1979; Rose, 1982a). Studies have indicated macrophages, granulocytes, and other leukocyte sub-populations are attracted to the lamina propria during primary infections, suggesting that these cells have a modulatory role in the intensity of Eimeria infections (Jeurissen et al., 1996). It appears that sporozoite contact with the mucosal surface activates a series of defense mechanisms, which lead to a local inflammatory cell influx. While much research has focused on lymphocyte involvement in protective immunity to Eimeria, other immune cell responses, particularly that of mast cells, to these pathogens have been neglected. Infection with Eimeria parasites produces reactions similar to those seen in infections with other parasites, both protozoa and helminths (Rose and Hesketh, 1982). However, there is limited data regarding the role of mast cells in the response to coccidial infection in poultry.Mast cells contain metachromatic-staining granules that store numerous inflammatory mediators. These cells originate in bone marrow haemopoietic tissues and migrate via the blood stream to the localized area where they develop and multiply. Mast cells are most commonly recognized for mediating the pathophysiology of allergic diseases (Abraham and Arock, 1998). However, evidence supports a multifaceted and significant role in immune reactions including mucosal inflammation, tissue repair, and other immunological responses (Yong, 1997). Mast cells possess distinct attributes that support their role in immune responses such as: 1) their location at the interface between the host and environment, in particular, around blood vessels, in the skin, and in mucosal surfaces including the
--------------------------------------------------------------------------------
Page 14
14gastrointestinal tract, 2) their capacity to release a wide range of inflammatory mediators, and 3) their capacity to undergo multiple cycles of mediator release (Abraham and Arock, 1998). However, researchers are limited in their ability to accurately describe the presence of mast cells during inflammatory responses due to changes in staining properties and morphological shape with respect to tissue location as well as variation between animal species (Yong, 1997).It is now known that in mammals, mast cell responses, especially their IgE mediated responses to certain parasitic infections, contribute to adaptive immunity (Rose, 1982a; Gray, 1976; Abraham and Arock, 1998). Mast cells accomplish this protection by releasing a myriad of pharmacologically active products including histamine, heparin, and serine proteases once the cell is stimulated by antigen, activation of IgE, or other host-derived proteins, which are generated during inflammatory reactions (Abraham and Arock, 1998). These mast cell derived products are considered to play a role in the innate rejection system of the host by promoting local smooth muscle contraction, an increase in vascular permeability resulting in localized edema, an increase in mucosal permeability, facilitating a rapid accumulation of antibodies and effector cells to mucosal surfaces, and intestinal ion secretion, in particular Cl-associated with intestinal anaphylaxis (Shi et al., 2000; Tizard, 2000). In studies conducted with mutant mice, virtually lacking mast cells, the specific importance of mast cell mediators was shown to be crucial in acquiring protective immunity to intestinal protozoal infections, specifically Eimeria infection (Rose and Hesketh, 1982; Huntley et al., 1985). In response to challenge with E. separata in rats, Shi et al. (2000) reported an increase in mucosal mast cells following primary challenge and a further stimulation of mast cell numbers by re-infection. Sensitization of the intestinal mucosa with antigen, especially in a secondary infection or exposure, results in the association of antibodies with mast cells in the lamina propria of the small intestine, which results in further stimulation of mast cell proliferation and subsequent release of their pre-formed granules (Harari et al., 1987; Harari and Castro, 1989). This rapid response of mast cell proliferationand activation results in changes of the physiologic environment in the intestine, altering the host-parasite interaction, and limiting, if not prohibiting the organisms ability to establish itself in the hosts tissue (Harari et al., 1987). The capacity of mast cells to induce
--------------------------------------------------------------------------------
Page 15
15pathological effects by excessive or inappropriate release of inflammatory mediators may contribute to an increased secretory response via excessive fluid and electrolyte secretion resulting in edema of intestinal tissue and ultimately, diarrhea. In addition, the quantity of mucosal mast cells responding to antigenic stimulation can dictate the intensity of the hosts physiological response (Harari and Castro, 1989). Such reactions could contribute to detrimental affects similar to what occurs with food allergies, including fecal blood loss, malabsorption, and villus atrophy (Metcalfe, 1984). While research has confirmed that mast cells play a major role in parasitic nematode infections in birds (Gray, 1976) and mammals (Rose and Hesketh, 1982b; Fernando, 1982; and Huntley, 1985), there is limited documentation regarding mast cell involvement in chicken Eimeria infections. Data reported by Rose and Hesketh (1982), Fernando (1982), and Huntley (1985), suggest similar roles of mammalian and chicken mast cells in enteric Eimeria infections. Rose et al. (1975) found that a hypersensitivity reaction, as shown by increased permeability to macromolecules of the gut, to EA parasites does exist. Affected duodenal tissues showed signs of edema with a thickened and juicy appearance due to responses mediated through the release of histamine mediators produced by an increase of mucosal mast cells. This response was greater in immunized animals when compared to controls, with a heightened response during secondary exposure to EA challenge. Similar studies have reported an acute intestinal mucosal mastocytophilia response to secondary Eimeria challenge in chickens (Rose et al., 1980; Daszak et al., 1993); however, it was questionable if there was a true increase in mast cell numbers or if cell migration occurred (Daszak et al., 1993). Although these reports describe the appearance of mast cells in enteric parasitic infections, there has been limited research to further characterize their specific effector roles in immune response mechanisms. Despite the evidence on a variety of immune responses induced in chickens by infection with coccidial parasites, further research needs to be conducted on accessory cells and intrinsic immune factors to aid in the development of control strategies.Immunovariability of the Eimeria Species Commercial broiler chickens are reared under conditions favorable for the propagation of coccidial infections. Historically, the severity of coccidiosis has been
--------------------------------------------------------------------------------
Page 16
16controlled through the continuous use of anticoccidial drugs and antibiotics, which have been used to reduce the frequency of apparent clinical signs and prevent widespread disease. However, coccidial outbreaks occur more often due to the rising emergence of drug-resistant strains. Since the late 1940s, producers have included prophylactic medication in the rations of broilers from one-day old until slaughter, which has resulted in widespread drug-resistance (Chapman, 1982). The efficacy of these drugs has been sequestered due to parasite modifications, which allow them to flourish in the presence of these preventatives. Until recently, coccidia were classified according to morphological, physiological, and behavioral characteristics such as those described by Brackett and Bliznick (1950). Today, it is known that different isolates within species of Eimeria exist (Chapman, 1982) but, little information is available comparing the immunogenicity and immunovariability of such isolates. Shirley (1985) described new techniques for revealing definitive markers of different strains within the same species of Eimeria; however, the differential host response to the different strains was discussed in minute detail. Several authors briefly state events within their experiments in which different strains were noticed (Talebi and Mulcahy, 1995). It has been shown that different strains are capable of having antigenic diversity, so that the immunity conferred by one species strain does not completely protect chickens against further challenge with a different strain of the same species (Talebi and Mulcahy, 1995). In a review on the use of coccidial vaccines by Danforth and Augustine (1989), it was stated that isolates from different geographical areas do not always show cross-protection following oral immunization. This may be a challenging factor in producing specific vaccines for coccidial control. More studies need to be conducted to evaluate the variability in the hosts protective immune response to different strains. This will expand the ability to find new alternatives for vaccine control; however, it is possible that the coccidia may find new ways to alter their antigenicity (Danforth and Augustine 1989).
--------------------------------------------------------------------------------
Page 17
17INTESTINAL MUCOSAL MAST CELL IMMUNE RESPONSE AND PATHOGENESIS OF TWO EIMERIA ACERVULINA ISOLATES IN BROILER CHICKENS
--------------------------------------------------------------------------------
Page 18
18INTRODUCTIONIn the modern commercial poultry industry, increases in disease and infections by enteric pathogens are common due to intense rearing systems. One disease of particular concern is coccidiosis. Coccidiosis is caused by species-specific protozoal parasites of the genus Eimeria. Infections caused by coccidial parasites have had a major economic impact on the commercial broiler industry in the past several decades. It has been reported that the US poultry industry suffers in excess of one to two billion dollars in annual losses relating to coccidial treatment, infection, and prevention (Danforth and Augustine, 1989; Talebi and Mulcahy, 1995; Yun et al., 2000). Chickens are infected by coccidial parasites through the fecal-oral route, and immunity is achieved once the parasite completes its life cycle within the host. Once the coccidial oocyst is ingested, Eimeria parasites penetrate enterocytes along the digestive tract at specific regions of the gut depending on the parasite species. Initial invasion is followed by subsequent parasite development, and severe damage to the intestinal lining occurs. This disruption of the mucosa leads to decreased nutrient absorption, increased feed conversion ratios, decreased weight gains, lethargy, diarrhea, and in severe cases, mortality. Historically, coccidial parasites have been controlled through the use of in-feed coccidiostats. However, through the years, drug-resistant strains of Eimeria have emerged, which hinder the efficacy of the presently used coccidiostats. Coccidial infections induce a variety of pathological and immunological responses which help the host defend against the parasite and acquire protective immunity. In addition, it has been reported that both immunologically mediated and non-immunological defenses play a role at the intestinal mucosal surface during Eimeria invasion (Lillehoj and Trout, 1993). It has been difficult to completely understand these protective mechanisms due to increases in drug-resistant strains and the complexity of the Eimeria species life cycle. Past studies have measured the severity of coccidiosis on the basis of weight gains, lesion scores, cessation of total oocyst output, and humoral and cellular responses (Stiff and Bafundo, 1993; Talebi and Mulcahy, 1995). However, a closer look at the histological and physiological changes at the mucosal level is pertinent to understand the specific nature of this immunity.
--------------------------------------------------------------------------------
Page 19
19It is well established that B and T-lymphocytes are involved in responses to Eimeriainvasion, but there is limited data concerning the possible participation of other effector cells, such as mast cells, which may participate in protective immunity. It is known that mast cell responses contribute to adaptive immunity in mammalian parasitic infections (Rose, 1982; Abraham and Arock, 1998), but their involvement in chickens has been largely overlooked. However, an acute mucosal mastocytophilia response has been reported during secondary Eimeria challenge in chickens (Rose et al., 1980). These responses have been questioned as to if there was an increase in mast cell numbers or if cell migration occurred (Daszak et al., 1993). Mast cells possess distinct attributes that support their role in immune responses, but researchers have been limited in their ability to accurately describe the presence of mast cells during inflammatory responses due to changes in staining properties and morphology associated with tissue location and species variation (Yong, 1997). If mast cells are in fact responsible for aiding in protective immunity, it is imperative that they are positively identified. Microscopic differential counting should indicate significantly elevated numbers of mast cells in the small intestine.Until recently, coccidia were classified according to morphological, physiological, and behavioral characteristics such as those described by Brackett and Bliznick (1950). Today, it is known that different isolates within species of Eimeria exist (Chapman, 1982). However, little information is available comparing the immunogenicity and immunovariability of different strains within each Eimeria species. Shirley (1985) described new techniques for revealing definitive markers of different strains within the same species of Eimeria; however, the differential host response to the different strains was discussed in minute detail. Several authors briefly state events within their experiments in which different strains were noticed (Talebi and Mulcahy, 1995). It has been shown that different strains are capable of having antigenic diversity, so that the immunity conferred by one species strain does not completely protect chickens against further challenge with a different strain of the same species (Talebi and Mulcahy, 1995). In reviews on the use of coccidial vaccines by Danforth and Augustine (1989) and Danforth (1998), it was stated that isolates from different geographical areas do not always show cross-protection following oral immunization. This may be a challenging factor in producing specific vaccines for coccidial control. Additional studies need to be conducted to evaluate the variability in the hosts protective immune
--------------------------------------------------------------------------------
Page 20
20response to different strains. This will expand opportunities for the development of alternative control measures; however, it is possible that the coccidia may find new ways to alter their antigenicity (Danforth and Augustine, 1989; Danforth, 1998). The interaction between the Eimeria and the intestinal mucosal immune system is a key component to the defense of the chicken to these enteric pathogens. Before control strategies can be improved upon, it is important to first understand the host-parasite interaction and the possible role of effector cells in the immune response. The aim of the present study was to further investigate the host-parasite interaction and the mechanisms responsible for protective immunity to coccidial parasites at the mucosal level in broiler chickens and to also determine if differential responses are associated with breed type. In the experiments conducted, two isolates of Eimeria acervulina (EA) were given to broilers of two breeds to determine if immunovariability exists between different strains within the samespecies and to analyze the differential mast cell and morphological intestinal host response with each. MATERIALS AND METHODSExperimental AnimalsIn all experiments, straight-run, day-of-hatch broiler chicks of breed A (Cobb) or breed B (Ross) were obtained from a local commercial hatchery. Chicks were placed on either clean pine shavings (Experiment 1, 2, 3, and 5) or wire-floored starter batteries (Experiment 4) and given ad libitum access to water and un-medicated starter feed, which met or exceeded NRC (1994) requirements. For the first two weeks of life, all chicks were housed in the same room with a controlled environment. Ambient temperature was set at 33°C for bird placement and decreased to 28°C by day 14. Supplemental heating lamps were provided for the birds during the first 5 days of life. At 14 days of age (day 15 for Experiment 3), chicks were weighed, wing-banded for breed and number identification, and randomly assigned to non-challenged or challenged treatment groups. Control birds were then housed in a separate room, within the same building, from the challenge birds for isolation purposes. Rooms were monitored and maintained at the same ambient temperature (28°C) from day of challenge (day 14 of age) through day 6 post-challenge (PC). In all
--------------------------------------------------------------------------------
Page 21
21experiments, rooms provided adequate negative pressure ventilation and birds were kept on continuous incandescent lighting. EimeriaTwo EA isolates, Eimeria acervulina isolate 1 (EA1) and Eimeria acervulina isolate 2 (EA2) were supplied by Dr. Harry Danforth (USDA/ARS, Beltsville, MD) for use in these experiments. Oocysts were stored at 4°C in a 2% potassium dichromate solution prior to challenge. Aliquots of the stock concentrations were taken at the time of each experiment and washed for removal of potassium dichromate prior to dilution to challenge inocula concentration. Briefly, oocysts for challenge were centrifuged for 10 minutes at 4°C and 845 x g. The supernate was removed, and the remaining pellet was re-suspended in ddH2O and centrifuged again using the same method. This process was repeated a minimum of three times to remove the potassium dichromate. After the final centrifugation, the pellet was re-suspended with sterile PBS for quantification of oocysts with a hemocytometer. Additional PBS was added to bring the inoculum to the desired challenge dose. Challenge concentrations for each experiment were determined by dose titration prior to the first trial and by the previous experimental results for all subsequent trials. All chicks in challenge groups received 1 mL of quantified sporulated oocysts per os directly into the crop using an oral gavage on day of challenge.Morphometric AnalysisDescending duodenal loop samples (25mm) were collected six days PC for analysis of cellular infiltrate of mast cells and morphological changes including villus height and crypt depth. Duodenal tissue from each broiler was fixed in 10% neutral buffered formalin for 48 hours and then placed in 70% ethyl alcohol (EtOH). Each tissue was cut into five sections for placement on each slide. Tissues were processed, embedded, and sectioned at 4 microns for slides. Tissues were stained with toluidine blue for the identification of mast cells and morphological measurements. Mast cell counts were made in the lamina propria region of the villus using an intraocular 5 x 5 eyepiece grid (total area 156.25 µm2) with the 40X objective of a bright-field microscope. A total of three complete tissues/slide and 15 grids/tissue were counted to quantify mast cells. Morphological changes were measured in millimeters using a bright-field microscope and the Sigma Scan Pro
software program11SSPS Inc., Chicago, IL
--------------------------------------------------------------------------------
Page 22
22(Figure 1). A total of three tissues/slide and four measurements/tissue were taken for changes in villus heights and crypt depths. Villus to crypt ratios were determined by averaging villus height measurements divided by crypt depth measurements.Experimental DesignFive experiments involving a total of 663 chicks were conducted to evaluate the intestinal response to two isolates of EA. In four of the five experiments, birds were challenged with one of two EA isolates, and data was collected to compare differences in body weight gain, lesion scores, morphometric alterations, and mast cell influx into the upper small intestine. The fifth experiment was conducted to determine if the differential host response was due to the parasite-host interaction itself, or if there was some other organism involved, such as a bacterial or fungal contaminant. In two of the experiments conducted, the responses of two commercial broiler breeds were compared to determine if there was a different degree of susceptibility or pathological response to the two isolates of EA.In all experiments conducted, birds were identified with metal wing bands (excluding Experiment 1) and weighed individually on day of challenge and six days PC for determination of body weight gain during the challenge. On day 6 PC, chicks were killed by cervical dislocation, and the duodenum of the upper small intestine was quickly removed and evaluated for intestinal lesion scores using the method described by Johnson and Reid (1970).Experiment 1. Straight-run broiler chicks of breed A (n = 105) were randomly divided and placed into two floor pens (n = 52 and 53/pen) for the first 14 days of life. On day 14, birds were randomly assigned to one of three treatment groups (n = 35/pen); control (non-challenged), EA1, or EA2. Birds were challenged per os with 1.25 x 105oocysts/mL of EA1 and 1.50 x 105oocysts/mL of EA2, respectively. On day 6 PC, all birds were weighed, lesions were scored (n = 35/treatment) and duodenal samples (n = 10/treatment) were taken for morphometric analysis.Experiment 2. Straight-run broiler chicks of two commercial breeds (breed A and breed B; n = 75/breed) were raised in two floor pens dependent on breed for the first 14 days of life. On day 14 (day 0 PC), birds were randomly separated into three treatment groups with an equal number of breed A and B (n = 25 birds per breed/treatment) within each group. Both challenge doses were increased by 2.5 x 104oocysts/mL upon evaluation of the
--------------------------------------------------------------------------------
--------------------------------------------------------------------------------
Page 2
2Our laboratory is particularly interested in the mechanisms responsible for mucosal immunity to coccidial parasites in broilers. Understanding these mechanisms has been complicated with the immunovariability associated with different isolates within the same Eimeria species. The purpose of the present study was to examine two different isolates of Eimeria acervulina and the pathology and immunovariability associated with both. Eimeria acervulina was chosen for the series of experiments because of its prevalence and continual occurrence in the commercial poultry industry. We hypothesized that immunovariability does exist between different strains within the same species of Eimeria and that there is a differential host response associated with each. In addition, mast cell numbers are increased during an Eimeria infection and exhibit effector functions, which mediate inflammatory responses and aid in intestinal immunity.
--------------------------------------------------------------------------------
Page 3
3REVIEW OF LITERATUREEimeriaCoccidiosis is a self-limiting, infectious disease of the digestive tract caused by host-specific intracellular protozoal parasites of the genus Eimeria. Coccidia are classified under the subkingdom Protozoa of the phylum Apicomplexia (Jeurissen et al., 1996; Lillehoj, 1996). As a group, coccidia of the genus Eimeria cause the most widespread health problems in the broiler industry and remain one of the most expensive diseases of commercial poultry production (Edgar, 1992; Henken et al.,1994; Yun et al.,2000). Birds infected with coccidial oocysts do not perform as well as non-infected birds as a result of moderate to severe damage to the intestinal mucosa. Birds exhibit decreased weight gains, increased feed conversion, and in some cases, birds may appear asymptomatic, but are limited in their ability to maximize feed efficiency. According to Edgar (1992), it takes only one viable oocyst to establish the presence of coccidia in a poultry house. This is possible because of the parasites reproduction index. Ingestion of one Eimeria acervulina (EA) oocyst in the infective state has the ability to yield about 72,000 oocysts in one complete life cycle (Henken, 1994). The prevalence of coccidiosis is worldwide and can be found in almost every commercial poultry flock (McDougald and Reid, 1997; Cox, 1998). In poultry, there are seven species of Eimeria that infect chickens (Shirley, 1986; McDougald and Reid, 1997). All of the chicken coccidia are pathogenic; however, some species produce more severe effects than others, such as severe morbidity and mortality. These microscopic, one-celled parasites invade their host via the fecal-oral route, and immunity is achieved once the parasite completes its life cycle in the host (Brackett and Bliznick, 1950; McDougald and Reid, 1997). Chickens of all ages are susceptible to coccidiosis, but birds that are three to five weeks of age are the most vulnerable (Edgar, 1992; Yun 2000). The Eimeria species have an extremely complex life cycle comprising stages both internal and external to the host. Infection occurs by ingesting sporulated coccidial oocysts found in contaminated litter, soil, feed or water. After ingestion, the protozoa go through a series of intracellular, extracellular, asexual, and sexual stages to produce viable oocysts that are excreted in the feces (Rose, 1987). After a brief period outside of the host, the oocysts become infective again through the process of sporulation, and the life cycle is complete.
--------------------------------------------------------------------------------
Page 4
4The impacts of coccidiosis are significant; however, eradication is impractical due to protective mechanisms of the oocysts. Eimeria possess a thick outer wall that acts as a protective barrier, which enhances the chance of survival under severe conditions. The oocysts are able to remain infective outside of the host for long periods, and their protective properties allow them to be resistant to many harsh chemicals and disinfectants (Eschenbacher et al., 1996; Jeurissen, 1996; Yun et al., 2000).Coccidian Life CycleAs the Eimeria species tend to be very specific in the intestinal region at which they invade, their life cycles are similar with a degree of species specificity. The Eimeriacomplete their life cycle in three distinctive phases including sporogony, merogony, (schizogony) and gametogony; however, the lengths of these phases are unique to the species (Yun et al., 2000). The following life cycle is adapted from Edgar (1992). Coccidiosis, caused by the ingestion of microscopic oocysts, is easily transmissible by mechanical means such as contaminated footwear and equipment, or it can be found in litter, contaminated soil, feed, or water (Conaway and McKenzie, 1991; McDougald and Reid, 1997). Chickens canbecome infected by coccidia once the oocysts develop into an infective stage outside of the host. Sporogony is the process of a one-celled zygote within the oocyst undergoing a series of divisions to form sporozoites, which are contained within sporocysts. Only oocysts that have undergone this process are able to cause disease. Sporulated oocysts contain four sporocysts, and each sporocyst contains two sporozoites. Mechanical action of the gizzard and pancreatic enzymes such as trypsin and bile salts cause the destruction of the oocysts outer wall, which releases the sporocysts into the digestive tract. The sporocysts are then further excysed by trypsin and bile salts that are present in the intestine. The sporozoites invade villus epithelial cells along specific locations throughout the digestive tract depending on the species of Eimeria. Some species travel within the mucosa, through the lamina propria to the crypt epithelial cells. Once inside villus or crypt cells, the process of merogony takes place. The sporozoite develops into a rounded body called a trophozoite, and then into an asexually reproductive first-generation schizont (meront). The schizont grows and divides rapidly to produce many first-generation merozoites. The bodies rupture and release hundreds of first-generation merozoites, which
--------------------------------------------------------------------------------
Page 5
5seek out and invade other epithelial cells. Second-generation trophozoites develop into second-generation schizonts. When the second-generation schizonts mature and rupture, increased quantities of invasive merozoites are released causing widespread infection. The number of asexual stages and time required for each depends on the Eimeria species involved; however, most species will have less than four asexual reproductive generations (Rose, 1987; Edgar, 1992). Merozoites produced by the latter schizont generations develop into sexual forms called gametocytes, some male and some female. This phase of sexual reproduction is termed gametogony. The female gametocyte matures into a macrogamete and the male gametocyte matures and ruptures releasing a large number of motile, biflagellate microgametes. The microgametes penetrate the mature female macrogamete and fertilization occurs. Following fertilization, a thickened protective wall forms around the zygote. At this stage, the zygote is considered an immature oocyst. When mature, the oocysts rupture the host cell, enter the lumen, and are expelled into the feces. Clinical signs are associated with tissue destruction from the release of the merozoites and mature oocysts from the mucosal surface during the last generations of merogony and throughout gametogony. In severe infections, much of the mucosal epithelium is sloughed off and nutrient absorption is compromised (Jeurissen, 1996; McDougald and Reid, 1997; Yun, 2000). Oocysts excreted from the birds remain in the environment and have the potential to infect other birds. Under favorable environmental conditions (approximately 84°F), sporulation of oocysts will be achieved in 24 to 48 hours, and the cycle will continue (Edgar, 1992; Graat et al., 1994; McDougald and Reid, 1997). According to Edgar (1992), once the oocyst is sporulated, it can remain infective to birds from several months to one or two years if protected from very hot, dry, or freezing conditions. Coccidial oocysts have rigid characteristics but are not totally indestructible. According to Lee (1988), unsporulated oocysts are more susceptible to physical and chemical agents than sporulated oocysts perhaps due to a highly sensitive metabolic state. Other factors, such as ammonia and anaerobic conditions, are also lethal to oocysts in the environment. Cessation of development occurs when oocysts are introduced to high levels of CO2or NH4,or exposed to mercury salts, mercuric cyanide, and mercuric chloride due to the ability of these agents to penetrate the oocyst wall (Kheysin, 1972). The prepatent period, the time it takes for oocysts to be seen in
--------------------------------------------------------------------------------
Page 6
6the feces after ingestion, is approximately 4 to 7 days for the Eimeria species (Henken et al., 1994; Jeurissen, 1996; McDougald and Reid, 1997). Coccidiosis in Commercial Broiler Operations Coccidiosis has plagued the poultry industry since the early 1900s, and by the 1940s, considerable information was gathered on the pathophysiology, immunology, epidemiology, and therapy of this disease. Large-scale production of poultry was developed, and with it came a need to study the control of threatening diseases (Brackett and Bliznick, 1950). Today, much knowledge has been gained about coccidiosis, but it continues to have a major economic impact on the commercial poultry industry. It has been reported that the US poultry industry suffers in excess of one to two billion dollars in annual losses relating to coccidial infection, treatment, and prevention (Danforth and Augustine, 1989; Talebi and Mulcahy, 1995; Yun et al., 2000). However, it is difficult to accurately estimate the total monetary losses suffered by the worlds poultry industry resulting from coccidiosis and its prevention or control (Danforth, 1998; Williams, 1999), because coccidiosis infects any type of poultry in any type of facility and its occurrence is worldwide (McDougald and Reid, 1997). Chicken coccidial species are highly species specific, and acquired immunity can be achieved once the coccidia complete their life cycle. However, birds can harbor the disease and be carriers after infection, increasing the likelihood of spreading coccidiosis (Lee and Shih, 1988; Williams, 1998). Most infections are mild due to the ingestion of few oocysts, and the disease will go unnoticed. With such infections, maximal feed efficiency and feed utilization by the bird is only slightly decreased. Ingestion of millions of oocysts, on the other hand, may cause severe infections, and a disastrous outbreak could occur. Flocks infected as a result of mild to severe exposure usually show a marked decrease in food and water consumption, and birds become depressed and tend to huddle. Decreased weight gains occur as a result of the disruption of the intestinal mucosa where minimal absorption is taking place. Diarrhea may result as the host is trying to flush the organism from the body, which may induce dehydration. Lesions of the intestinal mucosa and loss of pigmentation may also become apparent during the latter stages of infection (Conaway and McKenzie, 1991; Edgar, 1992; Lillehoj and Trout, 1993; McDougald and Reid, 1997).
--------------------------------------------------------------------------------
Page 7
7Mortality could result due to the lack of adequate nutrient intake, secondary infections, and other continual stressors associated with the diseased state. However, with the use of control agents such as medications, vaccines, or coccidiostats, the effects of coccidiosis are suppressed or even prevented. Since the 1950s, the control of coccidiosis has been achieved through anticoccidial compounds administered in the feed, which prevent or reduce infections to a sub-clinical level (Danforth, 1998). When used correctly, these compounds provide sufficient disease control. However, current management practices, such as intensive confinement, encourage the severity and transmission of coccidiosis. With the use of continual feed application of anticoccidials, unavoidable drug resistance by avian coccidia has resulted. This has allowed for an increased selection of drug-resistant strains, which reduce the efficacy of many anticoccidials that are used today. To further control the emergence of drug-resistant strains, shuttle and rotation programs have been pursued. These strategies have given good results in the past but continued efficacy is questionable. To date, the most serious limitation to anticoccidial therapy is the parasites increasing tolerance (McDougald and Reid, 1997). New and innovative anticoccidial products are unlikely to be marketed due to the increased costs of approving such compounds for food animals and consumer perspectives concerning feed additives (Danforth, 1998; Vermeulen, 2001). With such constraints, producers and researchers have tried to identify drug-free alternatives to coccidiosis control such as improved sanitation upon complete litter cleanout, selective breeding for improved immunity, and vaccination programs (Williams, 1998). However, developments of new and improved vaccines in recent years seem to have the greatest potential. In order for a commercial vaccine to be successful, it must have a reasonable cost, be as effective as other methods, give solid protection within a short period of time, and offer long-lasting immunity (Danforth and Augustine, 1989). To date, there are four vaccines available for commercial chicken flocks but their use in broilers is relatively low due to the short grow out period (Williams, 1998). The main objective of the broiler industry is to produce a high quality product at relatively low costs. Any extemporaneous costs related to treatment or prevention of enteric diseases increase production costs and lower profits. The drastic impacts on production caused by coccidiosis have significant economic effects at each facet of the industry. The
--------------------------------------------------------------------------------
Page 8
8goal of researchers and scientists is to sequester the effects of this enteric disease, which will in turn lower the overall costs of coccidiosis. Understanding the biological aspects of the host response to Eimeria infections at a cellular level is crucial to the development of new approaches to coccidia control.Eimeria acervulina In the poultry industry, nine species of the genus Eimeria have been described in chickens; however, in recent years, only seven of these species are known to exist and cause pathogenic effects (Conway and McKenzie 1991; Williams, 1998). Coccidial species are classified based on attributes such as: 1) intestinal location of infection, 2) appearance of intestinal lesions, 3) oocyst size, shape, and color, 4) average prepatent period length, 5) size of invasive sporozoites and merozoites, and 6) type of cell parasitized and location within epithelial cells. Based on these characteristics, E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox, and E. tenella are considered pathogenic for chickens. Of the seven species, E. acervulina (EA) has been one of the most frequently encountered coccidial parasites infecting poultry in commercial operations (Henken et al., 1994b; McDougald and Reid, 1997). Henken et al. (1994a) assumes that every flock is infected with coccidial parasites, and EA is responsible for the majority of these cases. Within the commercial broiler industry, infection with EA has major impacts on production. These impacts are related to how the host reacts to the parasite infection. However, there are other factors affecting the severity of disease such as the amount of viable EA oocysts ingested and pathogenicity of the isolate as well as the genetic, nutritional, and immune state of the bird (McDougald and Reid, 1997). A mild infection with EA could produce sub-clinical effects such as reduced feed utilization and increased feed passage, which can increase feeding costs and overall feed conversion ratios. However, a moderate to severe infection with EA can produce clinical signs such as loss of appetite, decreased water consumption, reduced weight gains, loss in skin pigmentation, diarrhea, lethargy, poor feed conversion, and whitish-gray lesions of the intestinal mucosa. Heavy infections may cause lesions to coalesce and death may occur (Johnson and Reid, 1970; Edgar, 1992; McDougald and Reid, 1997). While each species of Eimeria tend to be site specific, EA invades the anterior portion of the small intestine, specifically the duodenal loop and in severe infections, the anterior portion of the
--------------------------------------------------------------------------------
Page 9
9jejunum. It is speculated that secreted molecules from the invasion site itself and surface molecules located on the parasite could be responsible for this site specificity (Lillehoj and Trout 1993; Jeurissen et al., 1996).Immunity to Coccidial ParasitesAs described above, Eimeria reside outside the host for part of their life cycle but, the majority of it is completed inside the host during asexual and sexual stages of development occurring inside or outside enteric tissues. Once the bird ingests the viable oocyst(s), a cascade of events occur involving both non-specific and specific defense mechanisms of immunity (Lillehoj and Lillehoj, 2000). It is to be expected that the mechanisms responsible for immunity are complex due to the complexity of the parasite life cycle. Despite all of the research completed on immunity to Eimeria, no clear picture has emerged as to how complete resistance is acquired and which mechanisms are sequentially involved in generation of immunity (Rose et al.,1979; Danforth and Augustine, 1989). In naïve chickens, those previously unexposed to Eimeria, coccidial infections induce a variety of pathological and immunological responses, which help the host defend against the parasite and acquire protective immunity. However, the level of protection each facet of the immune system provides may vary with the developmental stage of the parasite (Rose, 1987). Prior to the generation of a specific immune response, the host tries to exclude the Eimeria through non-specific immune pathways such as competitive exclusion by normal flora, lysozymes, increased gastric secretions, and peristalsis to quickly flush parasites from the digestive tract (Lillehoj and Lillehoj, 2000; Yun et al., 2000). However, it has been reported that these innate defenses as well as specific immunologically mediated defenses play a role at the intestinal mucosal surface during Eimeria invasion (Lillehoj and Trout, 1993). Therefore, the naïve host probably does not eliminate the parasite utilizing only non-specific pathways, but infection can be controlled to a certain degree prior to the completion of the Eimeria life cycle and generation of a specific immune response.It has been determined that Eimeria parasites are vulnerable to the host immune system at three distinct phases of their development: 1) the period between excystation and sporozoite penetration of epithelium, 2) once the sporozoite enters the host epithelium and is exposed to intra-epithelial lymphocytes, and 3) during transport of the sporozoite from the
--------------------------------------------------------------------------------
Page 10
10surface enterocyte, through the lamina propria and into the crypt epithelium. After these stages in the life cycle, direct interaction between the parasite and cells of the host immune response are unlikely, and the probability of intervention is ceased (Jeurissen et al., 1996). During phases of the Eimeria life cycle that allow close contact with the immune system, both humoral and cell-mediated responses are stimulated; however, the contributions of each to protective immunity are still debated. The immune response to coccidial parasites has been extensively reviewed (Lillehoj and Trout, 1996; Yun et al., 2000). As discussed in these reviews and in other literature, the humoral arm of the immune system has been shown to play a role in conferring immunity to coccidial parasites, possibly more in the control of primary infections, as evidenced by studies with bursectomized and bursal-diseased chickens. It was shown that B-cell depleted animals are less resistant to primary infections and more susceptible to challenge than controls (Rose and Hesketh, 1979; Rose, 1987; Lillehoj and Trout, 1996). It has been suggested that species-specific circulating antibodies released into the lumen of infected birds have a protective effect by directly blocking invasion at the mucosal surface or by enhancing intraluminal destruction of sporozoites (Lillehoj and Trout, 1996). Serum from immune birds has also been shown to have a protective effect against coccidiosis when given to naïve birds (Rose, 1984; McDougald, 1985; Rose, 1987; Lillehoj and Trout, 1996; Yun et al., 2000). Although studies have shown that antibodies prevent initial invasion, it is less certain if they limit the course of disease once infection is established (Lillehoj, 1991). However, it is believed that cell-mediated immunity has a far greater role in protection against coccidial parasites than humoral immunity, but both systems are important for birds to acquire complete protective immunity (Rose, 1987; Jeurissen et al., 1996). Cell-mediated responses are an integral part of protective immunity as shown in experiments where T-cell depleted animals were unable to resist primary or secondary challenge infections (Rose and Hesketh, 1979; Rose, 1982a; Rose, 1987). Similar results were found when cyclosporin A (Cs-A), betamethasone, and dexamethasone, cell-mediated immune response suppressing agents, were given to naïve birds prior to Eimeria challenge (Lillehoj and Trout, 1993; Lillehoj and Trout, 1996). Talebi and Mulcahy (1995), observed a significant negative correlation between cellular lymphocyte responses and fecal shedding of oocysts in birds infected with homologous coccidia challenge, which supported the major
--------------------------------------------------------------------------------
Page 11
11role of cell-mediated immunity. Further studies have confirmed that T-cell sub-population numbers in the intestinal epithelium and lamina propria change during primary and secondary EA challenge, specifically, CD4+ T-cell increases during primary challenge and CD8+ T-cell increases during secondary exposure (Lillehoj and Trout, 1996). These results suggest that variations in T-cell sub-populations may reflect infection-related changes in the intestine. Martin et al. (1995) separated several fractions from sporozoites and merozoites in order to identify antigens from EA and how they contribute to protective immunity. It was determined that the synonymous effects of cell-mediated and humoral responses confer a species-specific protection, which lasts throughout the life of the bird. Protective immunity has normally been measured by maintenance of body weight gains, reduction in lesion scores, cessation in total oocyst output, and antibody and cellular responses in broiler birds (Stiff and Bafundo, 1993; Talebi and Mulcahy, 1995). While birds that have acquired protective immunity to a particular species of Eimeria no longer exhibit clinical signs of disease, they may continue to shed oocysts in the feces (Lee and Shih, 1988; Williams, 1998), a condition known as coccidiasis. However, Stiff and Bafundo (1993) showed conflicting results in a series of experiments that proved complete immunity can and does exist when birds are continuously challenged on a daily basis with homologous Eimeriachallenge. Nevertheless, protective immunity hinders fecal oocyst production, and invasion of the mucosal epithelium is altered. Interestingly, in immune birds, sporozoites penetrate the villus epithelium but are incapable of reaching the crypt epithelium and prevented from further development (Rose et al., 1984; Jeurissen et al., 1996).Physiological ChangesA closer look at histological and physiological changes that occur during the course of the immune response to coccidial parasites is pertinent to understand the specifics of immunity. Responses to Eimeria parasites at the intestinal mucosa may be mediated directly by the parasite or by the hosts immunoinflammatory response, which may result in changes in the intestinal mucosal morphology (Barker, 1993). Physiological changes related to infection have been well documented in experiments with EA infected chickens. Results indicate that at the level of the intestinal mucosa, local and systemic responses to coccidia are mediated in different ways. Fernando and McCraw (1973) measured the intestinal response
--------------------------------------------------------------------------------
Page 12
12to a single dose of EA, and infected birds showed a marked increase in total mucosal thickness, decreased villus height, and increased crypt length in the duodenum and to a lesser extent in the anterior jejunum. The mucosal alterations were the most severe at the height of infection, and a marked increase in the rate of replacement of intestinal epithelial cells was observed. In addition to an increase in duodenal epithelial cell turnover, Allen (1983) noticed an increase in metabolism of mucosal cells in the lower intestine, which may enhance compensatory growth and help the bird overcome the negative affects of infection on parameters of production. Other morphological changes were evident, such as increased gut length and increased tissue moisture (edema) in the intestine, which were not associated with starvation, but the sole effects of challenge. Changes in gut environment are also evident from challenge, such as decreased pH of the infected area (Ruff, 1984) and increased feed passage time (Stephens et al., 1974; McKenzie et al., 1986). These changes in gut homeostasis may alter feed intake, which leads to decreased nutrient digestion and absorption, changes in metabolism, and overall decreases in weight gains (Adams et al.,1996). Interestingly, researchers have contrasting results in experiments where weight gains are measured. Stephens et al. (1974) and Adams et al. (1996) reported decreased weight gains associated with EA challenge, while Ogbuokiri and Edgar (1985) and McKenzie et al. (1986) showed no significant weight gain differences when compared to non-challenged controls. This difference in results could be associated with several factors such as genetics of the birds, route of challenge administration, amount of infective oocysts administered for challenge, and strain of EA (Danforth and Augustine, 1989). Additional studies have shown that initial contact of the sporozoites with the intestinal mucosa produces an inflammatory response including marked cellular infiltration at the site of infection (Rose et al., 1979). This infiltrate consists of multiple leukocyte sub-populations, macrophages, natural killer cells, granulocytes, and lymphocytes that could modulate and enhance immune responses; therefore, altering nutrient absorption and decreasing weight gains (Jeurissen et al., 1996).The specific mechanisms by which each coccidial species causes disease is not clear; however, the establishment of secondary infections due to the altered intestinal mucosa may be responsible for a differential host response (Barker, 1993). This is possible because coccidia are able to interact with other pathogens such as bacteria and viruses, which may
--------------------------------------------------------------------------------
Page 13
13amplify the observed effects (Ruff, 1993). As stated above, the ability of the chicken to control the severity of infection and develop protective immunity to coccidial parasites depends on numerous factors. Host responses to the parasite involve a complex series of internal factors that are dependent on developmental stage of the parasite, immune status of the bird, and species and strain of the Eimeria parasite.Mast Cell Role in Coccidial ImmunityCurrent research has focused on alternative explanations for the mechanisms responsible for protective immunity to coccidial parasites. It is well established that many cells are involved in the response to Eimeria invasion, including rapid infiltration of polymorphonuclear cells (PMN), lymphocytes, and large mononuclear cells (LMN) to the site of inflammation (Rose et al., 1979; Rose, 1982a). Studies have indicated macrophages, granulocytes, and other leukocyte sub-populations are attracted to the lamina propria during primary infections, suggesting that these cells have a modulatory role in the intensity of Eimeria infections (Jeurissen et al., 1996). It appears that sporozoite contact with the mucosal surface activates a series of defense mechanisms, which lead to a local inflammatory cell influx. While much research has focused on lymphocyte involvement in protective immunity to Eimeria, other immune cell responses, particularly that of mast cells, to these pathogens have been neglected. Infection with Eimeria parasites produces reactions similar to those seen in infections with other parasites, both protozoa and helminths (Rose and Hesketh, 1982). However, there is limited data regarding the role of mast cells in the response to coccidial infection in poultry.Mast cells contain metachromatic-staining granules that store numerous inflammatory mediators. These cells originate in bone marrow haemopoietic tissues and migrate via the blood stream to the localized area where they develop and multiply. Mast cells are most commonly recognized for mediating the pathophysiology of allergic diseases (Abraham and Arock, 1998). However, evidence supports a multifaceted and significant role in immune reactions including mucosal inflammation, tissue repair, and other immunological responses (Yong, 1997). Mast cells possess distinct attributes that support their role in immune responses such as: 1) their location at the interface between the host and environment, in particular, around blood vessels, in the skin, and in mucosal surfaces including the
--------------------------------------------------------------------------------
Page 14
14gastrointestinal tract, 2) their capacity to release a wide range of inflammatory mediators, and 3) their capacity to undergo multiple cycles of mediator release (Abraham and Arock, 1998). However, researchers are limited in their ability to accurately describe the presence of mast cells during inflammatory responses due to changes in staining properties and morphological shape with respect to tissue location as well as variation between animal species (Yong, 1997).It is now known that in mammals, mast cell responses, especially their IgE mediated responses to certain parasitic infections, contribute to adaptive immunity (Rose, 1982a; Gray, 1976; Abraham and Arock, 1998). Mast cells accomplish this protection by releasing a myriad of pharmacologically active products including histamine, heparin, and serine proteases once the cell is stimulated by antigen, activation of IgE, or other host-derived proteins, which are generated during inflammatory reactions (Abraham and Arock, 1998). These mast cell derived products are considered to play a role in the innate rejection system of the host by promoting local smooth muscle contraction, an increase in vascular permeability resulting in localized edema, an increase in mucosal permeability, facilitating a rapid accumulation of antibodies and effector cells to mucosal surfaces, and intestinal ion secretion, in particular Cl-associated with intestinal anaphylaxis (Shi et al., 2000; Tizard, 2000). In studies conducted with mutant mice, virtually lacking mast cells, the specific importance of mast cell mediators was shown to be crucial in acquiring protective immunity to intestinal protozoal infections, specifically Eimeria infection (Rose and Hesketh, 1982; Huntley et al., 1985). In response to challenge with E. separata in rats, Shi et al. (2000) reported an increase in mucosal mast cells following primary challenge and a further stimulation of mast cell numbers by re-infection. Sensitization of the intestinal mucosa with antigen, especially in a secondary infection or exposure, results in the association of antibodies with mast cells in the lamina propria of the small intestine, which results in further stimulation of mast cell proliferation and subsequent release of their pre-formed granules (Harari et al., 1987; Harari and Castro, 1989). This rapid response of mast cell proliferationand activation results in changes of the physiologic environment in the intestine, altering the host-parasite interaction, and limiting, if not prohibiting the organisms ability to establish itself in the hosts tissue (Harari et al., 1987). The capacity of mast cells to induce
--------------------------------------------------------------------------------
Page 15
15pathological effects by excessive or inappropriate release of inflammatory mediators may contribute to an increased secretory response via excessive fluid and electrolyte secretion resulting in edema of intestinal tissue and ultimately, diarrhea. In addition, the quantity of mucosal mast cells responding to antigenic stimulation can dictate the intensity of the hosts physiological response (Harari and Castro, 1989). Such reactions could contribute to detrimental affects similar to what occurs with food allergies, including fecal blood loss, malabsorption, and villus atrophy (Metcalfe, 1984). While research has confirmed that mast cells play a major role in parasitic nematode infections in birds (Gray, 1976) and mammals (Rose and Hesketh, 1982b; Fernando, 1982; and Huntley, 1985), there is limited documentation regarding mast cell involvement in chicken Eimeria infections. Data reported by Rose and Hesketh (1982), Fernando (1982), and Huntley (1985), suggest similar roles of mammalian and chicken mast cells in enteric Eimeria infections. Rose et al. (1975) found that a hypersensitivity reaction, as shown by increased permeability to macromolecules of the gut, to EA parasites does exist. Affected duodenal tissues showed signs of edema with a thickened and juicy appearance due to responses mediated through the release of histamine mediators produced by an increase of mucosal mast cells. This response was greater in immunized animals when compared to controls, with a heightened response during secondary exposure to EA challenge. Similar studies have reported an acute intestinal mucosal mastocytophilia response to secondary Eimeria challenge in chickens (Rose et al., 1980; Daszak et al., 1993); however, it was questionable if there was a true increase in mast cell numbers or if cell migration occurred (Daszak et al., 1993). Although these reports describe the appearance of mast cells in enteric parasitic infections, there has been limited research to further characterize their specific effector roles in immune response mechanisms. Despite the evidence on a variety of immune responses induced in chickens by infection with coccidial parasites, further research needs to be conducted on accessory cells and intrinsic immune factors to aid in the development of control strategies.Immunovariability of the Eimeria Species Commercial broiler chickens are reared under conditions favorable for the propagation of coccidial infections. Historically, the severity of coccidiosis has been
--------------------------------------------------------------------------------
Page 16
16controlled through the continuous use of anticoccidial drugs and antibiotics, which have been used to reduce the frequency of apparent clinical signs and prevent widespread disease. However, coccidial outbreaks occur more often due to the rising emergence of drug-resistant strains. Since the late 1940s, producers have included prophylactic medication in the rations of broilers from one-day old until slaughter, which has resulted in widespread drug-resistance (Chapman, 1982). The efficacy of these drugs has been sequestered due to parasite modifications, which allow them to flourish in the presence of these preventatives. Until recently, coccidia were classified according to morphological, physiological, and behavioral characteristics such as those described by Brackett and Bliznick (1950). Today, it is known that different isolates within species of Eimeria exist (Chapman, 1982) but, little information is available comparing the immunogenicity and immunovariability of such isolates. Shirley (1985) described new techniques for revealing definitive markers of different strains within the same species of Eimeria; however, the differential host response to the different strains was discussed in minute detail. Several authors briefly state events within their experiments in which different strains were noticed (Talebi and Mulcahy, 1995). It has been shown that different strains are capable of having antigenic diversity, so that the immunity conferred by one species strain does not completely protect chickens against further challenge with a different strain of the same species (Talebi and Mulcahy, 1995). In a review on the use of coccidial vaccines by Danforth and Augustine (1989), it was stated that isolates from different geographical areas do not always show cross-protection following oral immunization. This may be a challenging factor in producing specific vaccines for coccidial control. More studies need to be conducted to evaluate the variability in the hosts protective immune response to different strains. This will expand the ability to find new alternatives for vaccine control; however, it is possible that the coccidia may find new ways to alter their antigenicity (Danforth and Augustine 1989).
--------------------------------------------------------------------------------
Page 17
17INTESTINAL MUCOSAL MAST CELL IMMUNE RESPONSE AND PATHOGENESIS OF TWO EIMERIA ACERVULINA ISOLATES IN BROILER CHICKENS
--------------------------------------------------------------------------------
Page 18
18INTRODUCTIONIn the modern commercial poultry industry, increases in disease and infections by enteric pathogens are common due to intense rearing systems. One disease of particular concern is coccidiosis. Coccidiosis is caused by species-specific protozoal parasites of the genus Eimeria. Infections caused by coccidial parasites have had a major economic impact on the commercial broiler industry in the past several decades. It has been reported that the US poultry industry suffers in excess of one to two billion dollars in annual losses relating to coccidial treatment, infection, and prevention (Danforth and Augustine, 1989; Talebi and Mulcahy, 1995; Yun et al., 2000). Chickens are infected by coccidial parasites through the fecal-oral route, and immunity is achieved once the parasite completes its life cycle within the host. Once the coccidial oocyst is ingested, Eimeria parasites penetrate enterocytes along the digestive tract at specific regions of the gut depending on the parasite species. Initial invasion is followed by subsequent parasite development, and severe damage to the intestinal lining occurs. This disruption of the mucosa leads to decreased nutrient absorption, increased feed conversion ratios, decreased weight gains, lethargy, diarrhea, and in severe cases, mortality. Historically, coccidial parasites have been controlled through the use of in-feed coccidiostats. However, through the years, drug-resistant strains of Eimeria have emerged, which hinder the efficacy of the presently used coccidiostats. Coccidial infections induce a variety of pathological and immunological responses which help the host defend against the parasite and acquire protective immunity. In addition, it has been reported that both immunologically mediated and non-immunological defenses play a role at the intestinal mucosal surface during Eimeria invasion (Lillehoj and Trout, 1993). It has been difficult to completely understand these protective mechanisms due to increases in drug-resistant strains and the complexity of the Eimeria species life cycle. Past studies have measured the severity of coccidiosis on the basis of weight gains, lesion scores, cessation of total oocyst output, and humoral and cellular responses (Stiff and Bafundo, 1993; Talebi and Mulcahy, 1995). However, a closer look at the histological and physiological changes at the mucosal level is pertinent to understand the specific nature of this immunity.
--------------------------------------------------------------------------------
Page 19
19It is well established that B and T-lymphocytes are involved in responses to Eimeriainvasion, but there is limited data concerning the possible participation of other effector cells, such as mast cells, which may participate in protective immunity. It is known that mast cell responses contribute to adaptive immunity in mammalian parasitic infections (Rose, 1982; Abraham and Arock, 1998), but their involvement in chickens has been largely overlooked. However, an acute mucosal mastocytophilia response has been reported during secondary Eimeria challenge in chickens (Rose et al., 1980). These responses have been questioned as to if there was an increase in mast cell numbers or if cell migration occurred (Daszak et al., 1993). Mast cells possess distinct attributes that support their role in immune responses, but researchers have been limited in their ability to accurately describe the presence of mast cells during inflammatory responses due to changes in staining properties and morphology associated with tissue location and species variation (Yong, 1997). If mast cells are in fact responsible for aiding in protective immunity, it is imperative that they are positively identified. Microscopic differential counting should indicate significantly elevated numbers of mast cells in the small intestine.Until recently, coccidia were classified according to morphological, physiological, and behavioral characteristics such as those described by Brackett and Bliznick (1950). Today, it is known that different isolates within species of Eimeria exist (Chapman, 1982). However, little information is available comparing the immunogenicity and immunovariability of different strains within each Eimeria species. Shirley (1985) described new techniques for revealing definitive markers of different strains within the same species of Eimeria; however, the differential host response to the different strains was discussed in minute detail. Several authors briefly state events within their experiments in which different strains were noticed (Talebi and Mulcahy, 1995). It has been shown that different strains are capable of having antigenic diversity, so that the immunity conferred by one species strain does not completely protect chickens against further challenge with a different strain of the same species (Talebi and Mulcahy, 1995). In reviews on the use of coccidial vaccines by Danforth and Augustine (1989) and Danforth (1998), it was stated that isolates from different geographical areas do not always show cross-protection following oral immunization. This may be a challenging factor in producing specific vaccines for coccidial control. Additional studies need to be conducted to evaluate the variability in the hosts protective immune
--------------------------------------------------------------------------------
Page 20
20response to different strains. This will expand opportunities for the development of alternative control measures; however, it is possible that the coccidia may find new ways to alter their antigenicity (Danforth and Augustine, 1989; Danforth, 1998). The interaction between the Eimeria and the intestinal mucosal immune system is a key component to the defense of the chicken to these enteric pathogens. Before control strategies can be improved upon, it is important to first understand the host-parasite interaction and the possible role of effector cells in the immune response. The aim of the present study was to further investigate the host-parasite interaction and the mechanisms responsible for protective immunity to coccidial parasites at the mucosal level in broiler chickens and to also determine if differential responses are associated with breed type. In the experiments conducted, two isolates of Eimeria acervulina (EA) were given to broilers of two breeds to determine if immunovariability exists between different strains within the samespecies and to analyze the differential mast cell and morphological intestinal host response with each. MATERIALS AND METHODSExperimental AnimalsIn all experiments, straight-run, day-of-hatch broiler chicks of breed A (Cobb) or breed B (Ross) were obtained from a local commercial hatchery. Chicks were placed on either clean pine shavings (Experiment 1, 2, 3, and 5) or wire-floored starter batteries (Experiment 4) and given ad libitum access to water and un-medicated starter feed, which met or exceeded NRC (1994) requirements. For the first two weeks of life, all chicks were housed in the same room with a controlled environment. Ambient temperature was set at 33°C for bird placement and decreased to 28°C by day 14. Supplemental heating lamps were provided for the birds during the first 5 days of life. At 14 days of age (day 15 for Experiment 3), chicks were weighed, wing-banded for breed and number identification, and randomly assigned to non-challenged or challenged treatment groups. Control birds were then housed in a separate room, within the same building, from the challenge birds for isolation purposes. Rooms were monitored and maintained at the same ambient temperature (28°C) from day of challenge (day 14 of age) through day 6 post-challenge (PC). In all
--------------------------------------------------------------------------------
Page 21
21experiments, rooms provided adequate negative pressure ventilation and birds were kept on continuous incandescent lighting. EimeriaTwo EA isolates, Eimeria acervulina isolate 1 (EA1) and Eimeria acervulina isolate 2 (EA2) were supplied by Dr. Harry Danforth (USDA/ARS, Beltsville, MD) for use in these experiments. Oocysts were stored at 4°C in a 2% potassium dichromate solution prior to challenge. Aliquots of the stock concentrations were taken at the time of each experiment and washed for removal of potassium dichromate prior to dilution to challenge inocula concentration. Briefly, oocysts for challenge were centrifuged for 10 minutes at 4°C and 845 x g. The supernate was removed, and the remaining pellet was re-suspended in ddH2O and centrifuged again using the same method. This process was repeated a minimum of three times to remove the potassium dichromate. After the final centrifugation, the pellet was re-suspended with sterile PBS for quantification of oocysts with a hemocytometer. Additional PBS was added to bring the inoculum to the desired challenge dose. Challenge concentrations for each experiment were determined by dose titration prior to the first trial and by the previous experimental results for all subsequent trials. All chicks in challenge groups received 1 mL of quantified sporulated oocysts per os directly into the crop using an oral gavage on day of challenge.Morphometric AnalysisDescending duodenal loop samples (25mm) were collected six days PC for analysis of cellular infiltrate of mast cells and morphological changes including villus height and crypt depth. Duodenal tissue from each broiler was fixed in 10% neutral buffered formalin for 48 hours and then placed in 70% ethyl alcohol (EtOH). Each tissue was cut into five sections for placement on each slide. Tissues were processed, embedded, and sectioned at 4 microns for slides. Tissues were stained with toluidine blue for the identification of mast cells and morphological measurements. Mast cell counts were made in the lamina propria region of the villus using an intraocular 5 x 5 eyepiece grid (total area 156.25 µm2) with the 40X objective of a bright-field microscope. A total of three complete tissues/slide and 15 grids/tissue were counted to quantify mast cells. Morphological changes were measured in millimeters using a bright-field microscope and the Sigma Scan Pro
--------------------------------------------------------------------------------
Page 22
22(Figure 1). A total of three tissues/slide and four measurements/tissue were taken for changes in villus heights and crypt depths. Villus to crypt ratios were determined by averaging villus height measurements divided by crypt depth measurements.Experimental DesignFive experiments involving a total of 663 chicks were conducted to evaluate the intestinal response to two isolates of EA. In four of the five experiments, birds were challenged with one of two EA isolates, and data was collected to compare differences in body weight gain, lesion scores, morphometric alterations, and mast cell influx into the upper small intestine. The fifth experiment was conducted to determine if the differential host response was due to the parasite-host interaction itself, or if there was some other organism involved, such as a bacterial or fungal contaminant. In two of the experiments conducted, the responses of two commercial broiler breeds were compared to determine if there was a different degree of susceptibility or pathological response to the two isolates of EA.In all experiments conducted, birds were identified with metal wing bands (excluding Experiment 1) and weighed individually on day of challenge and six days PC for determination of body weight gain during the challenge. On day 6 PC, chicks were killed by cervical dislocation, and the duodenum of the upper small intestine was quickly removed and evaluated for intestinal lesion scores using the method described by Johnson and Reid (1970).Experiment 1. Straight-run broiler chicks of breed A (n = 105) were randomly divided and placed into two floor pens (n = 52 and 53/pen) for the first 14 days of life. On day 14, birds were randomly assigned to one of three treatment groups (n = 35/pen); control (non-challenged), EA1, or EA2. Birds were challenged per os with 1.25 x 105oocysts/mL of EA1 and 1.50 x 105oocysts/mL of EA2, respectively. On day 6 PC, all birds were weighed, lesions were scored (n = 35/treatment) and duodenal samples (n = 10/treatment) were taken for morphometric analysis.Experiment 2. Straight-run broiler chicks of two commercial breeds (breed A and breed B; n = 75/breed) were raised in two floor pens dependent on breed for the first 14 days of life. On day 14 (day 0 PC), birds were randomly separated into three treatment groups with an equal number of breed A and B (n = 25 birds per breed/treatment) within each group. Both challenge doses were increased by 2.5 x 104oocysts/mL upon evaluation of the
--------------------------------------------------------------------------------
