Eimeria biological cycle: an example of perfect complexity in biology

The Eimeria biological cycle is a very complex one and is comprised of intracellular, extracellular, asexual and sexual stages. It is of paramount importance its understanding as its comprehension helps to understand the parasite epidemiology in the field, its pathogenicity and immunobiology.
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. The most commonly recognized species in the broiler chickens are Eimeria acervulina, E. tenella and E. maxima. E. praecox and E. mitis, whereas in layers and breeders it is likely to find also E. necatrix and E. brunetti seen that these two species need a longer period to replicate and reach a number of oocysts able to cause clinical signs and macroscopic lesions:

Gut localization of macroscopic lesions and categories of birds affected by the 7 Eimeria spp. affecting Gallus gallus.
The lifecycles of all these species of Eimeria are homoxenous and hosts are infected by the oral-faecal route. Chickens ingest sporulated oocysts, which contain four sporocysts, -each containing two sporozoites- from contaminated litter and then pass into the gizzard, where sporocysts excyst from the oocysts, thanks to the mechanic grinding of the gizzard together with the feed and its acid content. Subsequently the sporocysts reach the intestine, where – due to enzymes, bile and CO2 – they release the sporozoites which invade the cells of the intestinal wall. As we can see from the above table, each species of Eimeria has specific gut localization where it undergoes the schizogony causing different degrees of lesions depending on the seriousness of the infection. This is very important to keep in mind for diagnostic purposes.
Sporozoites of Eimeria brunetti and E. praecox penetrate the most superficial layers of enterocytes, whereas the sporozoites of other species (E. acervulina, E. maxima, E. necatrix and E. tenella) are able to reach the crypt epithelium, where they undergo development. Sporozoites penetrate into enterocytes’ core and start nuclear division followed by cytoplasmic differentiation. This replicative phase, called schizogony or asexual replication, leads to cellular damage in the epithelium, in fact ends with the formation of a mother cell, called schizont, with first generation of merozoites inside. The schizont grows, breaks the enterocytes and releases the first generation merozoites that penetrate other host cells. During the 2nd and 3rd phase of asexual replication gut damage becomes evident because of the high number of merozoites infecting enterocytes. The schizogony is important to understand the pathogenicity of each Eimeria spp., the immunobiology of the parasite (intracellular) and the way of attenuation of vaccines attenuated for precociousness. In fact, the strains contained in these vaccines undergo less schizogonies resulting in less gut damage with on the other hand the capacity to develop a full immunity.
After at least two generations of asexual reproduction, Eimeria merozoites enter the sexual replication or gamogony; in fact they invade the enterocytes and differentiate into either male (microgamonts) or female (macrogamonts). The microgamonts release many microgametes that exit, seek and fertilize the macrogamonts. This fusion produces a zygote (immature oocyst) that is subsequently excreted in the faeces. The gamogony is responsible of the passage of genetic information to the next generation of oocysts about resistance to anticoccidials.
Given the correct environmental conditions (warmth, oxygen and moisture), the oocyst sporulates and becomes infective undergoing sporogony (a meiotic process) that takes about 24 hours. The entire Eimeria cycle usually takes 4-7 days depending on the species.
This short life-cycle combined with the potential for massive reproductive capability during the intracellular phase, makes this group of parasites a serious problem under intensive farming condition.

This video explains the key points of Eimeria biological cycle and principles of immunology.


What are the pros and cons of changing from cycle vaccination to blanket vaccination in a PRRS stable farm?

Answered by: Enric Mateu Published on: May 27, 2016

According to the usual nomenclature1 farm is classified as stable if there are evidences of at least “a 90-day period of sustained lack of viremia in weaning-age pigs and no clinical signs of PRRS in the breeding herd“. In practical terms, this means that no viral circulation occurs in maternities and if there is viral circulation this is confined to other sections of the farm. A farm fulfilling these requirements is not free of the virus yet.
Vaccination against PRRSV is intended to reduce the clinical and economic impact of the infection and to reduce as well the transmission of the virus as much as possible. Bearing this in mind, to have a sows’ stock as homogeneous as possible in terms of the immunity against the virus is a desirable goal. In principle, blanket vaccination is more likely to produce that homogeneity than post-farrowing vaccination.
Certainly, if sows are vaccinated very close to the farrowing date with a modified live vaccine, the virus may eventually cross the placenta and produce the birth of viremic piglets. In principle, the practical impact of this with genotype 1 (European-type) vaccines, seem to be less important than with type 2 vaccines (North American type) although precise comparative data are lacking.

The so called 6-60 protocols were designed with two objectives: to provide (or sustain) immunity from the very start of gestation and to reinforce it before the critical phase for PRRSV transplacental infection avoiding vaccination of late gestation sows. This protocol of vaccination however does not produce better homogeneity than blanket vaccination and is being increasingly abandoned.

1. Holtkamp DJ, Polson DD, Torremorell M, et al. 2011. Terminology for classifying swine herds by porcine reproductive and respiratory syndrome virus status. J Swine Health Prod, 19:44:56

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New videotutorial on PRRSV Immunization: one of the key points for controlling PRRS on the farm

Introducing the fifth in PRRS Control recent series of educational videos that looks at immunization to help vets and producers immunize their breeding herds most effectively.

Proper immunization of animals is one of the key points of PRRS control because active immunization of animals aims to homogenize herd immunity status as well as confer proper cross-protection against the farm strain.

Before vaccination it is important to decide which is the target population to be vaccinated as well as the vaccine program to be implemented as there are different vaccine protocols. To decide on the vaccine program it is necessary to know the PRRS HERD STATUS as well as how good or bad the biosecurity of the farm is.

Vaccination is the best tool for immunizing animals and for providing protection against recirculation of the farm’s homologous strain as well as against the arrival of new heterologous strains on the farm

Coccidiosis in poultry: challenges for the industry

Coccidiosis in poultry is one of the diseases that probably cause most damage in the broiler rearing industry. It is not a case of producing high mortality within flocks but a subclinical process affecting economic performance, greater use of antibiotics and a loss of environmental farm conditions that will probably affect animal welfare.
Coccidiosis in poultry has become a truly complex disease that is not only an intestinal disease but also involves other issues that affect global production. In the past, coccidiosis was diagnosed as a clinical disease –even by the farmers- with coccidiosis caused by Eimeria tenella being the most simple example of this disease. Nowadays, and depending on the species of Eimeria involved, the disease can increase its complexity by producing not only a clinical process but also problems of global enteritis or even of immunosuppression.
From an economic perspective, the figure produced by the industry regarding the cost of coccidiosis in poultry is more than 1 billion US dollars.
Coccidiostasts have been used for a long time and, even applying a rotation strategy with different molecules, the risk of generating resistance is always present. We should take into account that, in fact, in recent years no new coccidiostasts have been launched on the market and we are working with the same old molecules to control coccidiosis in poultry.

E. praecox in the duodenum: A type of coccidiosis that is not clinical but causes a decrease in growth and feed conversion.
Improvements in avian genetics have developed broilers with a high growth rate and a lower rate of feed conversion. This means that the efficiency level is at a maximum, and together with this positive development, the quality of meat carcasses should be at the same high level. Nutrition plays a very important role in this but, more than this, intestinal integrity is essential in order to achieve maximum profitability of feed consumption.
Apart from these economic requirements, there are two important challenges for the industry: to meet welfare requirements and to reduce or even abandon the use of antibiotics. Under these circumstances, prevention is very important and, considering the potential ability for rearing today’s broilers, prevention of coccidiosis in poultry is a must.
The use of AGP (Antibiotic Growth Promoters) is a practice that was banned in Europe several years ago and is starting to be regarded with suspicion on other continents such as America and Asia. Nutrition companies have been looking for feed additives that could replace the effect of AGP apparently with good results but a proper strategy on nutrition is key: from raw material selection to appropriate physical presentation.
In this context, prevention of coccidiosis in poultry is regarded by companies as a major problem which must be tackled using the most appropriate means. Rotation with live attenuated coccidiosis vaccines is a strategy adopted by a large number of production companies all over the world. Rotation means using vaccines in 2 to 3 rearing cycles (Rotation program with coccidiosis vaccines) out of the total number of cycles annually. The purpose of this program is to maintain a low and sensitive population of oocysts on the farms, thereby minimizing the effects of coccidiosis in poultry.
There will probably be little room in future for the use of antibiotics or other substances regarded as antibiotics in the poultry industry. Now is the time to consider the use of vaccines for controlling coccidiosis in poultry.

What is the reliability of currently available diagnostic kits for detecting PRRS?

Answered by: Enric Mateu

In general it can be considered that the performance of diagnostic tests for PRRSV is good for detecting infected animals if a good sampling strategy is applied.
The performance of a diagnostic test is usually evaluated in terms of diagnostic sensitivity and specificity. Technically, diagnostic sensitivity is defined as the proportion of true infected (diseased) animals that are detected by the test (usually classified as positive) while diagnostic specificity is the proportion of healthy animals that are correctly classified as such (namely, negative). In general, diagnostic sensitivity depends on analytical sensitivity; namely, the lowest amount of analyte (i.e. antibodies or viral particles/genomes) that the test can detect.
Regarding serological tests for PRRSV (ELISA), most of them are able to detect infected animals between 7 to 14 days after the onset of the infection and detect antibodies for more than 4-5 months. After the second week of infection sensitivity can be considered close to 100% or 100%. Regarding specificity, most ELISAs show diagnostic specificities above 97-98%, that are quite acceptable. In our experience, the agreement of individual results between different commercial ELISAs is moderate-to-good in terms of classifying infected and non-infected animals.
However, the practical performance of a test should be evaluated not only with regards to sensitivity and specificity but also in consideration to the epidemiological context. As a concept, the use of a test that is not 100% sensitive in a population with a very low proportion of infected animals would lead to the inability to detect those animals (false negative). On the contraire, the use of a less-than-100% specific test in a healthy population, sooner or later will lead to the detection of false positive animals. Our current serological tests are very good for detecting infected animals and a little less good for classifying healthy animals.

In the case of PCR, the abovementioned concepts applied the same but some considerations are to be made. Although the analytical sensitivity is very high (it is possible to detect as little as 1 viral in the PCR tube) and the specificity is close to 100% the high genetic diversity of PRRSV affects the practical performance of the test. In other words, since the virus is so diverse it is almost impossible to design a PCR detecting all possible isolates within one genotype. In our laboratory, we did some trials to assess the potential of both commercial and in-house PCRs and the best ones detected about 95-97% of the isolates within genotype 1. This means, that occasionally, some infected animals can test negative just because the diversity of the virus. However, it is important to consider that most of the failures corresponded to isolates belonging of subtypes 2-4 of Eastern Europe.
A different subject is how reliable is a given result produced by a given laboratory. In order to assure the mentioned sensitivities and specificities is important that the laboratory have adequate protocols, including a quality control protocol, and participate regularly in ring trials or collaborative diagnostic exercises in order to test internally and externally the quality of the results.
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Oocyst vaccine production: A challenge for animal health companies

Prevention of coccidiosis in poultry relies on live vaccines. Ionophores act as coccidiostasts by maintaining a certain level of contact with the oocyst parasite but vaccines are the only products that are able to generate a good level of specific immunity from the very beginning. Oocyst production in itself is a challenge for those companies producing vaccines

Vaccines have been considered as a method of control of coccidiosis since the early 1950s when the first products appeared. In view of the complexity of the immunity established to fight against the disease, it is necessary to work with the infective part of the parasite, i.e. the sporulated oocyst. This has been the mode of action of coccidiosis vaccines. The first vaccines were produced from pathogenic oocysts gathered in the field. The oocysts were roughly processed to obtain a suspension containing a minimum quantity of sporulated oocysts. Obviously, at this time it was quite difficult to obtain a uniform number of these oocysts and the process of sporulation could not guarantee the number needed for complete immunization.

Microscopic picture (x400) of the oocysts of 5 species included in HIPRACOX®
The current systems of production of vaccines against coccidiosis are based on oocyst production in live birds. A certain number of companies are producing these oocysts in SPF birds (Specific Pathogen Free) to be sure that no maternal antibodies can cause interference in the process and to minimize microbiological or viral contamination. For those who are familiar with poultry, oocyst production could be considered similar to egg production but instead of eggs, oocysts are produced. So a farm with extremely strict biosecurity measures is needed.
The main challenge of a coccidiosis vaccine is the transformation of faecal material into a sterile product and not all the vaccines marketed worldwide achieve this. This means that the process must be strictly monitored from the beginning (including the incubation of the SPF birds) to the end of the process when the vial is filled with the oocyst suspension.
Vaccines are made of oocysts of different Eimeria species. The type of species depends on the level of protection required in short or long cycle birds. The number of oocysts per species depends on the strain of Eimeria selected. This is the reason why different vaccines with the same species of Eimeria can have different numbers of oocysts per dose.
In the 1990’s, a new generation of vaccines appeared on the market. These were vaccines produced with attenuated strains. An attenuated strain is produced from an oocyst modified to generate less impact on the intestinal mucosa with a better and earlier immune response (Attenuated strains in coccidiosis vaccines)
To produce vaccines with attenuated strains is a costly process due to the lower reproductive potential of these modified strains. However, the behaviour of these strains is totally different when comparedwith wild strains, with minimum impact on the intestinal mucosa and no impact on performance in birds.
Finally, the latest generation of vaccines includes not only oocysts but new adjuvants that are able to enhance specific cellular immunity. Based on the study of the substances that play a role in coccidiosis immunology, it is possible to identify those that are more important for activation of the cells involved in the global process and amplify their production. They could in fact be regarded as intelligent vaccines because not only do they trigger the immune system but they also help the global mechanism, especially in a disease in which cellular immunity plays an important part.

Coccidiosis in chickens: It’s time for vaccination

Coccidiosis in chickens is one of the more common and widespread diseases. Since the beginning of the industrial production of broilers, veterinarians and farmers have been trying to control this costly parasite.
The annual cost is believed to be around $1.5 billion/year. The cost of coccidiosis in chickens is based on direct production losses and indirect costs through the application of control measures.
In the past, the fight against the different Eimeria species was through the use of several molecules in the food. The first chemicals used against coccidiosis in chickens were introduced in the 1950’s. It soon became clear that there were some efficacy problems with these molecules, in the sense that the Eimeria spp. present on the farms could develop resistance against them after they were used consecutively a couple of times on the same farm. In fact, the very high potency of chemicals blocks parasite multiplication, but selection towards resistance is rapid. This is the reason why chemicals are used for shorter periods; in so-called “clean-up” programmes.
Then during the 1970’s a new generation of anticoccidials was launched: the ionophores. Ionophores always allow for a limited multiplication of parasites, known as “coccidial leakage”. Although this might seem to be a negative feature at first sight, it leads to slower development of reduced sensitivity. Over the years and after thousands of tonnes have been used, resistance has become an important problem and has developed to all compounds currently in use. More and more vets are asking for a solution.
The first vaccine appeared in 1952 in the US. Unfortunately, non-attenuated strains are used in this vaccine and some medication with anticoccidials is very often still needed after vaccination. These old vaccines are still in use in some countries today.
A new generation of vaccines against coccidiosis in chickens, using attenuated oocysts of Eimeria spp., has been launched in recent years:
HIPRACOX® vaccine was launched in 2008 in Europe and is still today, the only vaccine for broilers with Eimeria praecox. It also contains E. acervulina, E. maxima, E. tenella and E. mitis, making its composition ideal for broilers which very often suffer from subclinical coccidiosis in chickens.
Now HIPRA is launching EVALON®, a new Eimeria vaccine for long life birds (breeders and layers):
EVALON® contains E. acervulina, E. maxima, E. tenella, E. brunetti and E. necatrix. In these high value birds, clinical coccidiosis is one of the biggest concerns, because it is associated with increases in mortality, a decrease in uniformity and generally the increase of ∑ associated with intestinal damage or stress to the birds with consequent economic losses. Thus, the composition of the vaccine has to be studied with specific reference to these five most pathogenic species.
The vaccine vial comes in the same box with its solvent, HIPRAMUNE® T, which contains:
A dedicated colouring agent and a vanilla aroma that enhances the intake of the vaccine even under low light conditions.

An adjuvant which modulates the immune response, for the first time in a live vaccine.

As the Product Manager of EVALON® explains in this article, this new generation of adjuvanted live attenuated vaccines against coccidiosis in chickens will allow poultry veterinarians and producers to choose Eimeria Prevention using vaccination only.
It is a new era for the Prevention of coccidiosis in chickens. Now it is time for Eimeria Prevention with vaccines.

Can we observe acute clinical symptoms of PRRS in sows with low titers of antibodies against PRRSV? Answered by: Tomasz Stadejek.   Published on: May 12, 2016

Most serological analyses for PRRSV are made with ELISA. There are several ELISA kits on the market but most of them detect antibodies against nucleocapsid protein. This protein is being produced in large quantities in infected cells and stimulates strong seroconversion. However, these antibodies have no impact on protection against reinfection.
So, we can only interpret the seroconversion detected in ELISA as a proof of contact of a pig with PRRSV. Not as a proof of immunity against infection or clinical symptoms. The problem with serological assessment of sows using ELISA is that most of kits give some percent of false positive results. The phenomenon has been known for many years but the problem was not fully solved with new generations of ELISA kits.

Another aspect of interpreting ELISA results is that the method can be used for assessment of populations and not for individuals. After infection or vaccination antibodies start to appear at 10-14 days, the level grows for several weeks and then decreases. Some animals can become seronegative at 4-6 months post exposure.

It is dependent of the immunizing strain and also of the dose. In a stable sow herd (no virus circulation, no vertical transmission) gilts can be highly positive if immunized at quarantine, while old sows could be often low positive or negative. In unstable farm (virus circulates and is being vertically transmitted) naïve replacement gilts will seroconvert and may exhibit clinical symptoms. Small farms can spontaneously clear off PRRSV if internal replacement is applied.

If naïve replacement gilts are introduced, they will remain seronegative, while old sows can show some levels of antibodies. Answering the question, yes, low positive sows can show acute clinical symptoms of PRRS.

To be sure that the suspected symptoms are caused by PRRSV the best is to test fetuses or weak piglets by PCR. Detecting PRRSV will be an ultimate proof of PRRS caused reproductive failure. Serological analysis (unless it takes into account detailed analysis of multiple age sows) will be of little diagnostic help.

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Would you recommend the vaccination of a PRRS seropositive herd, even there are no clear reproductive and respiratory signs, or would you recommend specific management measures?

Answered by: Tomasz Stadejek I Published on: May 5, 2016

Not always we can observe clear clinical symptoms of PRRS. More often, in endemically infected farms, the disease affects the production parameters and its diagnosis can be made based on detailed analysis of those. If precise figures are not known or the losses are not evident it is good to evaluate the virus circulation, either with serological methods (ELISA) or PCR.
If PRRSV starts to circulate pre-weaning or early post weaning it is indicative for unstable reproductive herd. It means that there is vertical transmission from sows to piglets. Even if the losses in the reproduction are minimal (only gilts maybe affected) the price is paid in piglets. Not necessarily directly due to PRRSV but more often due to multifactorial infections as PRRSV modulates immune system. It would be strongly recommended to vaccinate replacement gilts at quarantine, and the reproductive herd, in order to eliminate vertical transmission and increase maternal immunity in piglets. If the virus starts to circulate later in nursery, vaccination of piglets could be considered.
It has to be kept in mind that the immunity against PRRSV is slow to develop. Piglets should be vaccinated at least 4 weeks before the infection is expected to occur. The efficacy of modified live vaccine in piglets can be compromised by maternal antibodies. The virus contained in such vaccine must be able to effectively replicate in vivo. High levels of maternal antibodies can neutralize the vaccine virus before it is able to properly stimulate the immune system of a piglet.
There are several management practices that could be applied together with, or instead of vaccination program, to break the infectious chain in a herd.Which method is best justified in a given farm situation has to be assessed individually, considering all the possible constraints regarding the implementation of the management changes, and their cost. So, the seropositive status of a farm is too unspecific to be able to decide for, or against vaccination.
The virus reservoir has to be known based on detailed ELISA or PCR analysis and then that action against it could be planned. Whether or not PRRSV control or elimination in endemically infected farm will bring significant improvement in production parameters depend on many factors besides PRRSV.
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