Coccidiosis in chickens: the role of subclinical species of Eimeria

The fight against coccidiosis in chickens means the adoption of different strategies depending of the type of bird. If we are managing long life cycle birds, we have to pay special attention to clinical Eimeria species that are able to generate a real coccidiosis process with macroscopic lesions and symptoms that will reduce the healthy status of the birds and will compromise the development of immunity against other diseases or cause the death of the birds.


However, when we are rearing standard, certified or even free-range broilers, the focus needs to be a different one. In these cases it will be difficult to find real clinical coccidiosis. Otherwise, the “silent” species – such as Eimeria praecox among others – will affect the intestinal mucosa and will reduce the capacity of a broiler for nutrient absorption. Dealing with subclinical species is essential in coccidiosis in chickens with a high growth rate.

For a long time, there was a trend to classify coccidiosis in chickens according to the age of the birds. It was considered that E. acervulina, E. maxima and E. tenella were species affecting all the birds. In the case of long life cycle birds such as breeders and layers, it was necessary to include E. necatrix and E. brunetti in the composition of vaccines against coccidiosis. Indeed, this is true for long life cycle birds because feed conversion and growth is important, but not the key point. In fact, these birds are reared under feed restriction.

The chicken intestine has a very important part that plays a main role in digestion and absorption of nutrients. This is the duodenum.
The duodenum is a part of the small intestine where the main digestive processes occur. The pancreas will provide digestive juices into the duodenum for protein digestion in particular and the liver (via the gall bladder) will produce the bile, a gastric juice involved in the digestion of lipids and absorption of vitamins A, D, E and K. When we are treating coccidiosis in chickens –and more specifically in fattening broilers- it is essential to identify the species of Eimeria located in this part of the intestine.
E. acervulina, one of the most prevalent species in broilers, prefers this part of the intestine even if the level of infection is moderate. It is quite typical to find the white ladder-like spot lesions scattered and confined to the duodenum:

E. praecox is also located in the duodenum. For a long time, E. praecox was considered to be a non-pathogenic strain. After the studies by Williams et al. (2009), the pathogenicity of E. praecox was demonstrated. There are two facts to consider with regard to the damage caused by E. praecox in coccidiosis in chickens:

On the one hand, praecox causes microscopic damage in the cells of the duodenum. In infections with E. praecox oocysts (106) it is possible to see villus atrophy, crypt hyperplasia and increased leukocyte infiltration.
On the other hand, praecox modifies the viscosity of the liquids in the duodenum. In infections with E. praecox an increase in whitish mucus and non-digested feed is observed.

Finally, in other studies carried out by Répétant et al. 2011, the impact of the infection caused by E. praecox was related to the infective dose but its impact on performance was present from the lowest dose of 5,000 oocysts/bird when it was co-administered with E. acervulina.

After all these arguments it seems of great importance to include Eimeria praecox as a target in the fight against coccidiosis in chickens in fattening birds where the integrity of the duodenum is a must. This is the reason why in the design of the composition of HIPRACOX® it was decided to include E. praecox together with E. mitis to protect against the effects of subclinical coccidiosis in chickens.

The Eimeria species responsible for coccidiosis in broiler chickens

The Eimeria species responsible for coccidiosis in the species Gallus gallus are: E. acervulina, E. maxima, E. mitis, E. praecox and E. tenella, which are responsible for the disease in short life-cycle poultry (broilers), and E. necatrix and E. brunetti, which, together with the above 5 species, are responsible for the occurrence of outbreaks in long life-cycle poultry (breeders and layers). They are all ubiquitous in their behaviour and vary in their pathogenicity.


There are seven Eimeria species that are responsible for avian coccidiosis, 5 of which cause the disease in broilers: E. acervulina, E. mitis, E. tenella, E. maxima and E. praecox.

There are two more strains of Eimeria that are not recognised as causing the disease. These are: E. hagani, the only description of which was by Levine P.P. in 1938; and E. mivati, a species found in a vaccine in the USA which appears to be a mixture of other Eimeria, E. mitis and E. acervulina (M. W. Shirley et al. 1983).
Focussing on the species that are really important in the generation of the disease in broilers, we need to know exactly which species cause most damage and how they interact by generating synergies between one another, thereby causing greater damage within the host. E. acervulina, E. maxima, E. tenella, E. mitis and E. praecox are the main species that cause avian coccidiosis in broilers. They can all be found along the intestine of poultry, affecting different areas and causing different lesions depending on the species concerned.
Focussing on E. acervulina, E. maxima, E. tenella, E. mitis and E. praecox, the species that are responsible for avian coccidiosis in broilers, we need to know the lesions they cause and their behaviour and distribution within the gastrointestinal tract of chickens. Below is a video giving details of the different species of Eimeria that affect the gastrointestinal tract of poultry.
For the development of vaccine strains, the Eimeria species have to be attenuated. There are three methods of doing this: passage through embryonated eggs, gamma irradiation and selection for precociousness. When they are selected by passage through embryonated eggs and by gamma irradiation, this is generally associated with a loss of immunity by the line and therefore stable attenuation is not maintained (Shirley M.W et al. 1984). The best method shown so far is selection for precociousness developed by Jeffers (1975). during the nineteen seventies. The method is based primarily on the reduction of the reproductive potential of the strain, resulting in attenuation of the virulence, maintenance of the immunogenicity and genetically controlled stability.
When the appropriate method of attenuating the Eimeria species has been identified, we need to know which species will be necessary for development of the vaccine. E. acervulina, E. maxima and E. tenella are well known as the pathogens responsible for the disease and are included in the great majority of existing commercial vaccines, but this is not the case with E. mitis and E. praecox, which have been regarded as species of “less importance” in the field.
In his study on the pathogenesis of Eimeria praecox in broilers, R.B. Williams et al. (2009) demonstrated the importance of E. praecox as a pathogenic strain in itself. In this study, it was compared with E. acervulina and it was observed that the lesions caused by E. acervulina were macroscopic and severe, but did not last for more than 14 days post-infection. In contrast, in the case of E. praecox, the lesions were microscopic and caused a reduction in the viscosity of the intestinal content. In other studies such as those carried out by J.M. Répérant et al. 2011, the impact of the infection caused by E. praecox was related to the infective dose but its impact in performances was present from the lowest dose of 5000 oocysts/bird. When it was co-administered with E. acervulina, it caused a greater impact on production indices.
Furthermore, the selection of strains within an Eimeria species is essential in order to obtain good protection, thus conferring cross-protection within each species, as is the case with the vaccine strain E. maxima 013, which is able to provide protection against 6 different pathogenic strains of E. maxima obtained from different geographic locations.
It is essential to find out about the epidemiological behaviour of the Eimeria oocysts in order to understand how the vaccine strains will behave and what vaccine load will be necessary in order to produce an appropriate vaccine response. Generally, the oocysts that are found in the bedding can persist for up to 3 weeks (Williams R.B. 1995), with sporulation being better with a moisture content of the litter from 31 to 62.1%. Whether or not they are sporulated, 20% are ingested by the chickens and pass through their intestines. As immunity is generated within the flock, the percentage of oocysts eliminated is reduced with each life cycle of the parasite. Hence the necessity of determining accurately the volume of oocysts necessary for each vaccine strain, in order to ensure an appropriate response to the vaccine.
In order to confer immunity against all the Eimeria species that are present in the environment, all the Eimeria vaccine strains need to be included as there is no cross-protection between the different species, hence the necessity of formulating a vaccine with the 5 Eimeria species when the target category is represented by broilers.
With all this tested knowledge, HIPRA, a specialist in avian coccidiosis, has developed the HIPRACOX® vaccine, the only vaccine specially developed for short life-cycle poultry, containing in its formulation all the Eimeria species responsible for avian coccidiosis in broiler chickens.

Eimeria tenella is probably the most diagnosed Eimeria on the planet, but what is the prevalence of the other Eimeria species that cause coccidiosis in poultry?

Eimeria tenella is by far the most widely detected species on farms when routine lesion scoring is performed. However, it is well known that Eimeria infections very seldom occur with one single species of Eimeria, most of the time they are multiple. Let’s investigate what are the most prevalent species and how multiple infections usually occur.


As Eimeria tenella is probably the easiest species to detect by lesion scoring, a common belief is that this species is the most prevalent all over the globe. In fact, macroscopic lesions are amongst the most pathognomonic with blood or typical moulds in the caecum and common finding of bloody droppings in the litter.

There are seven species of Eimeria recognized as parasitizing chickens (Gallus gallus), which vary in their ability to induce diarrhoea, morbidity and mortality (Williams 1998). They are Eimeria tenella, Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix and Eimeria praecox. They occur throughout the world wherever commercial broilers are reared. All seven species of Eimeria infecting chickens were detected in surveys of commercial poultry farms in many countries, for example, the Czech Republic (Kučera 1990), France (Williams et al. 1996), Sweden (Thebo et al. 1998), the UK (Eckert et al. 1995), Argentina (Mcdougald et al. 1997; Mattiello et al. 2000), Australia (Jorgensen et al. 1997; Morris et al. 2007), China (Sun et al. 2009), India (Aarthi et al. 2010), South Korea (Lee et al. 2010) and Brazil (Moraes et al. 2015).
Like Eimeria tenella that is localized in the caecum, the different Eimeria species tend to develop in different parts of the chicken gut and may be identified by the nature and location of the lesions they cause during multiplication (Long et al. 1976, Long et al. 1982). However, a definitive diagnosis requires additional laboratory investigations. Nowadays, polymerase chain reaction (PCR) and morphometric identification of the Eimeria species are frequently used together as a means of differentiation in the field samples of faeces and litter.
In Europe, few field surveys of Eimeria species are available and even fewer have been conducted using samples from broiler farms. In a study conducted by HIPRA (Pagès et al. 2015), litter samples obtained from broiler farms between 2003 and 2008 in Spain, Belgium, Italy and France were evaluated for the presence of Eimeria species. Samples of litter faeces from each farm were pooled from 10 different random locations within a single broiler house on each farm. In fact, the species composition of coccidial populations is highly repeatable among different broiler houses on the same farm (Jeffers 1974). The evaluation was performed using a polymerase chain reaction (PCR) developed at the Institute for Animal Health (Compton, UK) to specifically detect E. tenella, E. acervulina, E. maxima, E. mitis and E. praecox. Together with this molecular tool for detecting Eimeria species in litter samples, oocyst counts and the evaluation of the percentage of species by using a morphometry test were also performed to further evaluate the samples.
We decided only to look for the five species of Eimeria that usually affect commercial broiler farms -Eimeria tenella, Eimeria acervulina, Eimeria maxima, Eimeria mitis, and Eimeria praecox- due to the fact that E. necatrix has been reported to cause disease in long-lived birds -up to 12 weeks or more- (Williams et al. 1996, Williams 1998) and similarly E. brunetti is often reported to be rare in broilers (Long et al. 1982, Williams et al. 1996, Graat et al. 1998).
Analyzing the 3 species of Eimeria of known and high pathogenic potential (Eimeria tenella, E. acervulina and E. maxima), Eimeria acervulina has been shown to be the most widespread in the four European countries studied, whereas between the two species causing subclinical problems and affecting productivity: E. mitis seems to be quite uncommon, whereas E. praecox was shown to be present in all countries. Combinations of 3 species together were the most common especially: E. tenella, E. acervulina, and E. praecox. E. praecox was found to be highly associated with E. acervulina.


Similar to the study conducted in Europe and using the same techniques of evaluation of the samples, in 2012 HIPRA also performed the first Eimeria spp. prevalence study in South Africa (Pagès et al. 2015)


Analyzing the 3 species of Eimeria of known and highly pathogenic potential (Eimeria tenella E. acervulina and E. maxima) Eimeria acervulina was shown to be the most widespread in South Africa (40.5%), whereas regarding the 2 species that cause subclinical problems and affect flock productivity: E. mitis was less prevalent (7.1%) then E. praecox (9.5%). Combinations of 2 species together were the most common especially: E. acervulina + E. tenella and E. acervulina + E. maxima.
Once again these studies showed the widespread presence of Eimeria praecox and thus of subclinical coccidiosis that “remains one of the most important infections causing decline in production performances” (Haug et al. 2008).
Finally, these results confirmed that the most prevalent species of Eimeria by far is E. acervulina, in contrast with the field perception that most of the time coccidiosis is only caused by E. tenella.

What is a correct way to choose a PRRS sow vaccination program? When PRRS vaccination of piglets is needed?

Answered by: Enric Marco

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Piglet vaccination is usually done when we would like to reduce PRRS virus circulation on the growing phase. Vaccine will reduce the amount of PRRS virus shed by viremic animals and also will reduce duration of shedding. Those reductions are not spectacular but can help to control the PRRS virus circulation.
Sow PRRS vaccination is used in two ways. The first one is to bring back to normality the farm after an outbreak; the second one is to help the farm to maintain stability by avoiding PRRS virus recirculation among sows which will produce viremic piglets.
By vaccinating sows, immunity will be higher and more stable helping to reduce subpopulations of sows which we know are the responsible for on farm virus recirculation.

Eimeria praecox: a brief story of the big unknown of coccidiosis in poultry

Seven species of Eimeria (E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella) are recognized to be causative agents of coccidiosis in chickens of the genus Gallus gallus. Until recently, Eimeria praecox was considered to be a non-pathogenic species unable to cause adverse effects in the host. Read more

In fact, in 1970, when Johnson & Reid wrote the milestone article that for the first time standardized and described the scoring scale for lesions caused by all Eimeria spp., Eimeria praecox was not included. It was, and still is, well know that E. praecox is not able to provoke pathognomonic lesions like E. acervulina, E. brunetti, E. maxima, E. necatrix and E. tenella, however even then some researchers were investigating whether this species of Eimeria was truly non-pathogenic.

Peter L. Long in 1968, attempted to demonstrate the adverse effects of Eimeria praecox on the host and for this purpose he compared its effects with those of Eimeria acervulina. His conclusions were that even if E. praecox was less pathogenic than E. acervulina, high doses of oocysts of E. praecox depressed the rate of weight gain and food and water consumption.
Some years later, in 1982, Gore and Long reported that mortality and morbidity are not characteristics associated with Eimeria praecox, however in their studies it was shown to interfere with digestion and consequently cause significantly retarded weight gain. It was the first time that E. praecox was recognized to be economically significant and thus could no longer be considered non-pathogenic.
At the same time, the first prevalence studies were performed and the general outcomes for Eimeria praecox prevalence were quite surprising: this species was shown to be present in many countries all over the globe and with percentages that were far from what was considered to be negligible. See table here. 

From 2003 to 2008 HIPRA also performed several prevalence studies in Europe -namely in Spain, Belgium, Italy and France- and the widespread presence of Eimeria praecox was also confirmed by these (Pagès et al. 2015). 


In 2012 HIPRA performed the first study of Eimeria spp. prevalence in South Africa (Pagès et al. 2015) and there, too, E. praecox was found to be present on 9.5% of farms.
Further confirmation that Eimeria praecox can exhibit a wider range of virulence than previously thought, came in 2009 from a study conducted by Willians et al. where it was observed for the first time that E. praecox was able to cause actual body weight loss and market increases in FCR, as did E. acervulina. Also for the first time, it was observed that the virulence of E. praecox may not only be equal to but may exceed that of E. acervulina.


In the field, single Eimeria species infections are quite rare and most of the time infections involve multiple species. Since Eimeria praecox replicates in the same tract of the gut as E. acervulina -the duodenum- and both impact early in the production cycle, one of the aspects to be investigated was whether during a co-infection with these two species either a competitive exclusion or a synergic effect took place. In 2012, Répérant et al. found that when E. praecox -even with a low infective dose- is inoculated together with E. acervulina, its negative effect on growth was added to the latter and significantly increased compared to a single E. acervulina infection.
Nowadays it is quite clear that Eimeria praecox constitutes one of the major causes of subclinical coccidiosis with repercussions on the productive performances and due to its early impact in the cycle, it is especially important in broiler meat productions where the first weeks are crucial for the overall performance of the birds. It is therefore worth taking into account this species of Eimeria in prophylactic approach to fully protect poultry.
HIPRACOX® was the first attenuated vaccine for coccidiosis in broilers that took this aspect into consideration by also including E. praecox in its composition.

Coccidiosis in poultry: an objective assessment of the incorporation of a rotation programme using precocious attenuated vaccines

Within the scope of assessing new strategies for the control of coccidiosis in poultry, the first factors to consider are always immunological and physiological but also include less objective factors such as the management. However, when these new strategies come to be assessed, the exercise has to be carried out in perspective by evaluating those indicators that are the most critical in order to decide whether maintaining such strategies or to replacing them with others. In the production of broiler chickens, these indicators are solely productive.

Live precocious attenuated vaccines for coccidiosis in poultry like HIPRACOX® have been used in numerous countries and situations, and these experiences serve to support the implementation of vaccine rotation programmes for the control of coccidiosis in poultry.

Over the past 10 years, experiences in the field have provided data regarding this cost/benefit ratio, derived from the use of rotation programmes with precocious attenuated vaccines, such as HIPRACOX®. Having vaccinated several million birds in different circumstances throughout the world, these experiences have enabled us to draw various conclusions.
Minor intestinal injuries
Reduction of the total mean lesion score (TMLS), according to the Johnson and Reid method (1970) for coccidiosis in poultry, in vaccinated and post-vaccination cycles compared to pre-vaccination cycles (Dardi et al. 2015, see image above).
Improvements in production indicators
Production results improved during the vaccination phases and in subsequent cycles where the use of anticoccidial drugs was reincorporated, both in winter vaccine cycles in Spain (Alameda et al. 2015) and in central European farms during the summer (Ronsmans et al. 2015):

Cost-benefit assessment

Production costs per 1,000 kg were reduced, as a result of the birds’ improved performance.


(Alameda et al. 2015) showed that production cost (euros/1,000 kg live weight) was always below average (€923) in the months with vaccination cycles (CDV) compare with previous cycles with anticoccidial drugs (CAV). From the first cycle, there was a positive trend that persisted in all subsequent cycles (CPV). The cost included the value of the vaccine given on the first day of life by coarse spray.

Using precocious attenuated vaccines such as HIPRACOX® against coccidiosis in poultry provides several advantages over traditional programmes based solely on control through the use of anticoccidial drugs. Aimed at broiler chickens in particular, this control strategy is being used in several companies across different countries with optimum results. Ultimately, it has proven a simple tool for reducing the use of antibiotics, as indicated by the major producing countries.

How an adjuvant can modulate the immune response against coccidiosis in poultry

 

EVALON® is a live coccidiosis vaccine against avian coccidiosis in poultry composed of E. acervulina, E. brunetti, E. maxima, E. necatrix and E. tenella. All the strains have been selected to maximize immunogenicity. Avian Eimeria have a complex life cycle with a combination of exogenous and endogenous stages that trigger the immune system of the host. However, Eimeria parasites have also been described as being highly elusive to the immune system as well as producing chemokines than can slow or inhibit the immune response (Jang 2011, Schmid 2014, Miska 2013).

Although it is well known that live vaccines can induce an adequate immunity, we strongly believe that immune modulation is crucial in providing a strong, fast and long-lasting immunity (Dalloul 2005). This could be essential in the prevention of coccidiosis in poultry.

In a study conducted at the University of Zaragoza with Prof. Emilio del Cacho, different groups of birds received EVALON®, EVALON® together with HIPRAMUNE® T and PBS (control group).
HIPRAMUNE® T is a solvent specifically designed to be mixed together with the vaccine EVALON®. It contains a colorant and flavour -to enhance pecking and preening when the vaccine is applied in coarse spray administration- and an adjuvant designed to modulate the immune response. It is the first time that the use of an adjuvant has been considered in a live vaccine intended to prevent coccidiosis in poultry.
Birds from each subgroup were used to obtain intestinal lymphocytes from mucosa and Peyer’s patches at different times post-vaccination.
Results obtained in the first experiment indicated that HIPRAMUNE® T is able to increase the level of Th1 cytokines, as indicated by the results obtained for IL-2. Regarding IFN-gamma, statistically significantly higher levels were detected on different days both in the mucosa and Peyer’s patches. In contrast, levels of IL-4 and IL-10 were equal or lower when the group receiving EVALON® plus HIPRAMUNE® T was compared to the group receiving EVALON® alone. These results confirm the ability of HIPRAMUNE® T to stimulate a cellular immune response. It is therefore hypothesized that EVALON®, when administered together with HIPRAMUNE® T, is able to polarize the immune response towards a Th1 response. This happens with more intensity than the vaccine without the adjuvant. The Th1 response is crucial for protection against Eimeria (del Cacho 2011 and 2012).
In vaccines designed for layers and breeders which are long-lived categories, it is essential to have extended protection throughout the life cycle. Generally, live attenuated vaccines have proved to provide protection until 37 weeks. However, in the case of EVALON®, its efficacy is boosted by co-administration with HIPRAMUNE® T; we therefore wanted to test the duration of immunity until the end of the production cycle of a breeder hen (60 weeks).
In a second study, the duration of immunity was assessed for EVALON® plus HIPRAMUNE® T. The laboratory facilities for the performance of the trials prevented the introduction of external Eimeria oocysts which could provide trickle infections throughout the rearing and laying period. Together with this, birds were not moved from rearing to laying. It is well known that at farm level and after vaccination, trickle infections occur and it is important to maintain and enhance long-term immunity against Eimeria parasites (Williams 2002). In the present study we wanted to prove that protection was extended, even in the absence of trickle infections.
At day 0 a group of one-day-old birds was vaccinated via coarse-spray with one dose of vaccine EVALON® plus HIPRAMUNE® T while another group of birds received only PBS (control). The elimination of oocysts was monitored weekly in litter faeces, as can be seen in the Figure below. After the vaccination peak and once the birds became fully protected, generally no oocysts were detected.


To study the efficacy of the vaccine, birds were randomly selected at different time points (14, 28, 40 and 60 weeks) and individual challenge tests for each Eimeria species included in the vaccine were performed using highly pathogenic heterologous challenge strains. The vaccinated and non-vaccinated birds were compared, the main parameter under consideration being the macroscopic intestinal lesions after the challenge (Johnson & Reid 1970). Other secondary parameters evaluated also included individual body weight, elimination of oocysts post-challenge, clinical signs and mortality.
As an example, data obtained for E. necatrix lesion scoring after challenges is included in the Figure below. Similar results in terms of a significant reduction in lesions in vaccinated groups were obtained for all the other Eimeria species included in the vaccine.


The results obtained indicated an extended duration of immunity with EVALON® when administered together with the adjuvanted solvent HIPRAMUNE® T in conditions that do not favour the presence of oocysts in the litter. The duration of immunity was confirmed at 60 weeks post vaccination.

Immunology in coccidiosis in chickens: The role of cytokines IL-2 and IFN-gamma

The cellular and molecular mechanisms leading to immune protection against coccidiosis in chickens are complex and include multiple aspects of innate and adaptive immunity. Innate immunity is mediated by subpopulations of immune cells that recognize pathogen-associated molecular patterns. Adaptive immunity, which is important in conferring protection against secondary infections, involves subtypes of T and B lymphocytes that mediate antigen specific immune response. Experimental studies in coccidiosis in chickens now support the role of lymphocytes and their secreted products (Lillehoj et al. 2011)

Eimeria parasites have a long and complex biological cycle with exogenous and endogenous phases that trigger the immune system of the host. These parasites that cause coccidiosis in chickens can produce substances (chemokines) than can inhibit the immune response.

It is well-known that coccidiosis vaccines can generate a proper level of immunity but at HIPRA we worked on the hypothesis that if we were able to modulate the immune response in some way we would be able to generate a strong, fast and long lasting immunity (Dalloul et al. 2005).

In coccidiosis in chickens there are 2 types of immune response addressed by two types of cellular populations: those producing interleukins Th1 (IL-2 and IFN-gamma) and others that produce interleukins Th2 (IL-4 and IL-10). To enhance the control of the parasite through cytotoxicity mediated by antibodies it is very convenient to shift the immune response towards Th1without eliminating the synthesis of interleukins type Th2.
A Th1 immune response will improve the production of specific cells such as macrophages and Natural Killer cells (NK). Interleukins IL-2 and INF-gamma play an important role in this immune response. IL-2 is responsible for the evolution of the primary T-cells towards a Th1 response and IFN-gamma will allow a differentiation of the non-differentiated cells into macrophages and NK-Cells.
When a pathogen enters the bird, it is initially contained by physical barriers but if it is able to penetrate and successfully infect it, the innate immunity is going to take an active role. At this point in a primary infection, macrophages are very important. They will capture the pathogen, digest the proteins and present them. Together with macrophages, dendritic cells and B cells are specialized antigen presenting cells that will activate naïve T-cells. Antigen Presenting Cells in fact are seen as the link between innate and adaptive immunity. Once the adaptive immunity is created, this will provide a specific response that will eliminate the pathogen.
In the case of an intracellular pathogen, which is the case with Eimeria in coccidiosis in chickens, there will be an activation of IL-2 that will switch the response to a Th1 response. In the Th1 immune response, activated Cytotoxic T-cells (CTL) are able to detect infected cells and destroy them.
CTL will target schizonts and sexual stages of the parasite. This will produce IFN-gamma and activation of macrophages and NK cells. When this Th1 response is successful, the intestinal damage caused by the parasite stops and there is a dramatic decrease in the elimination of oocysts.
If, on the other hand, we have an extracellular pathogen, it is more convenient and effective to have antibodies against this pathogen. IL-4 will lead to a Th2 response. This is what the parasite wants because it is less effective; in fact, the Eimeria parasites have coded in their genome the right proteins to immune modulate the host to have this type of response. The B-cell response will increase IL-10 that will activate eosinophils, mast cells and basophils that are all important in an inflammatory process.
In the studies conducted by HIPRA we wanted to follow the production of Th1 cytokines (IL-2 and IFN-gamma) and Th2 cytokines (IL-4 and IL-10). In this way we could better understand all the mechanisms involved in this process and look for adjuvants that were able to modulate the immune response against coccidiosis in chickens.

What we have learned about PRRS disease after 20 years?

The Porcine Reproductive and Respiratory Syndrome has been the disease that has changed many ideas in global pig production in the same way as HIV did in the human population. Listening to the testimonies of opinion leaders in this video, it is fair to say that we have learned a lot and that the video captures two visions: that of the scientists and that of the practitioners.

Conceptually, there are certain similarities between the PRRS and AIDS, the latter appearing in the 1980s, whilst PRRS appeared in the 1990s. In both cases, they are very serious diseases in the affected populations and despite the fact that there is still no cure, there has been much progress in our knowledge of the viruses that are responsible for them, and how to improve control strategies for them. Improved biosecurity in pig production is equivalent to the educational measures used to reduce the risk of sexual transmission. 
Fortunately, today the mortality associated with HIV, despite having been very high, has been dramatically reduced and is associated with concomitant diseases, sounds familiar? 
There are also major differences between the two syndromes – the acquired immunodeficiency syndrome remains a potential cause of death throughout the world, with an estimated 37 million affected individuals* and, making a rapid calculation on the basis of a population of some 7,000 million, this means about 0.5% of the world population, and this is where it differs radically from the PRRS virus, as only 5% of the world pig population is considered to be free of the Porcine Reproductive and Respiratory Syndrome. 
If we look back to the first cases of both diseases, a great deal of time and money has been invested in deepening our understanding of both syndromes and in both cases there has been a great deal of progress so that today it is possible to live with both viruses and lead a dignified life as an AIDS sufferer or achieve an acceptable level of production if you are a PRRS-positive producer. 
Indeed, it is one of the lessons that our opinion leaders (the practitioners) have learned, we have learned to live with the disease, although there is still a great deal to learn because, as Alberto Stephano says, just when you think you know everything there is to know about the PRRS virus, you get PRRS again. The approach by our colleague Carlo Lasagna using an (Italian style) football metaphor is also worthy of note, firstly defence (biosecurity) and then good attacking (optimization of control measures). 
As for the scientists, we have learned how to diagnose it, monitor it, sequence it and even how it interacts with the immune system or how it evades it, we have learned how it can mutate or vary genetically, and a key area in which there has been a great deal of research (especially by American universities) has been the main routes of transmission of the disease (as with HIV, the greatest efforts have been devoted to minimizing transmission). 
Key aspects on which work is being carried out in the sector are biosecurity, both external and internal, and immunization etc., which should be understood as a series of measures, because if they are taken separately, the probability of failure increases exponentially. 
Following a more modern approach, Darwin Reicks points to air filtration for the control of aerosols and also, more recently, the probability of working with animals that are genetically resistant to the Porcine Reproductive and Respiratory Syndrome virus.

Contribution of DNA-based diagnostic methods to the treatment of coccidiosis in poultry. What’s New?

In the fight against infectious diseases, the first step is the correct identification of the causative agent, and the symptoms and lesions that it causes in the host. A correct diagnosis influences on the effectiveness of the treatment established, particularly if it is combined with preventive measures such as vaccination. Traditional techniques such as microscopic observation and oocysts counting remain very useful as screening methods, and an aid in the diagnosis and treatment of coccidiosis in animals. DNA-based methods such as PCR have overcome some limitations of these conventional methods, allowing the analysis of more samples in less time, increasing sensitivity and allowing the quantification of the parasite in one step.


These new methods positively influence the treatment of coccidiosis, expanding the possibilities for the poultry veterinarian to control the disease.

Diagnosis of coccidiosis in poultry by stool analysis methods in the laboratory has some limitations inherent to the type of sample used. Faeces collected on the farms are sent to the laboratory for microscopic examination of Eimeria oocysts. If this first step takes too long oocysts change their morphology, which makes microscopic identification tricky. The possibility of obtaining false results when analysing samples in poor condition is high. This may affect the effectiveness of the treatment of coccidiosis in broilers and laying hens.
Another limitation of conventional parasitology in the diagnosis of coccidiosis in poultry is the simultaneous presence of three or more species of Eimeria within a flock of birds. If the treatment of coccidiosis or its prevention by vaccination is aimed at the kind of acting Eimeria, misidentification could induce treatment failures. Finally microscopy as a diagnostic method of coccidiosis in poultry is limited to the identification of the species of parasite and the approximate amount present in a gram of sample. Other characteristics such as resistance to certain anticoccidials, etc., cannot be assessed by these techniques.
At the end of the 90s, new methods of detection and identification of Eimeria in chickens based on DNA techniques were developed. Specifically PCR (polymerase chain reaction) protocols for epidemiological purposes (Schnitzler 1998) were described. Subsequently the PCR method has been improved, and in the 2000s the first methods of real-time PCR (Kawahara 2008) that reduced analysis time, improve assay sensitivity and reduce cross-contamination between samples were optimized. This made possible to establish the treatment of coccidiosis more accurately and quickly.
The refinement of PCR methods has been continued in recent years. One of the limitations inherent to the analysed samples is the presence in the faeces of substances capable of inhibiting the action of the polymerase, which is the engine of the synthesis of DNA copies during the amplification step. The development of optimized methods of obtaining and purifying DNA (Gerhold 2015), and the addition of internal control (IPC) in each reaction well (Raj 2013), has largely reduced false negative results due to reaction inhibition. The IPC must be positive in each and every one of the samples analysed in order to be considered a valid PCR assay.
The PCR reaction can be divided into four phases that are displayed in the amplification plot (see image above).
More recently, the real-time PCR has been optimized for simultaneous obtaining of qualitative and quantitative results. Thus, it is possible to get accurate identification of the species of Eimeria present in the sample as well as the amount of each of them (Nolan 2015). At present, the PCR is a robust and target-specific detection method for Eimeria in poultry. The PCR solves some major limitations of microscopic examination of stool, allowing analysis of large numbers of samples in less time, the examination of faeces, tissues and even suspensions of oocysts, and even serves as a method to ensure the identity and purity of strains present in vaccine preparations (You 2014).
Therefore, the PCR has a direct impact on the treatment of coccidiosis, based on a timely and reliable diagnosis. However the laboratories using quantitative PCR methods for the diagnosis of coccidiosis in poultry, require highly trained personnel, calibrated instruments, and standardized protocols to ensure repeatability and accuracy.
Finally, successes in diagnostic and subsequent treatment of coccidiosis in poultry depend largely on the interpretation of the results. Interpretation should be based on the clinical history, the estimated time of infection and signs and lesions observed. PCR along with the classic method of observation of the parasite and the clinical judgment allows improving the treatment of coccidiosis on the basis of a comprehensive approach.
This video animation illustrates the main advantages of the quantitative real time PCR.