A Comparative Study of Different Immunoassays to Detect Specific Antibodies to Echinococcus Spp. in Human Sera.

Human echinococcosis, one of the most serious of parasitic zoonoses, is caused by the larval stages of taeniid cestodes of the genus Echinococcus. The study aimed to assess the reliability of the detection of specific antibodies to E. multilocularis and E. granulosus s.l. in human sera and to compare their diagnostic potential for their utilization in the practice. In the study, the somatic antigen of E. multilocularis (AgEm), antigen B (AgB), and the hydatid fluid antigen of E. granulosus and two commercial ELISA kits - Echinococcus granulosus (Bordier Affinity Products, Crissier, Switzerland) and NovaLisaTM Echinococcus IgG (NovaTec Immunodiagnostica, Germany) - were compared. Sera of patients with alveolar and cystic echinococcosis, and with different parasitic/other infections were used to evaluate the sensitivity, specificity and cross-reactivity of in-house and commercial ELISA methods. AgEm presented the highest values regarding the diagnostic indicators, showing 100 % specificity and 90.0 % sensitivity. The tests for serological diagnostics of cystic echinococcosis were less sensitive and specific. The Echinococcus granulosus kit had 83.8 % specificity and 88.2 % sensitivity, while AgB and AgHF showed 85.0 % and 86.3 % specificity, and 76.5 % and 100 % sensitivity, respectively. NovaLisaTM Echinococcus IgG proved to have 95.7 % specificity and 77.8 % sensitivity. The results point out that the combination of different serological tests and approaches in accordance with clinical and imaging findings is still essential to prove the correct diagnosis in suspected patients.

Seroprevalence of Trichinella infections in domestic dogs from Slovakia.

Trichinellosis is constantly present in the sylvatic cycle in Slovakia, with several sporadic human outbreaks registered since the 1960s. The largest outbreak of trichinellosis occurred in 1998 and was related to the consumption of dog meat that had been added to smoked pork sausages. The aim of this study was to estimate the seroprevalence of Trichinella infection in domestic dogs in various regions of Slovakia. Out of 439 dogs tested, 56 (12.8%) were classified as a seropositive based on the enzyme-linked immunosorbent assay (ELISA), with confirmation by Western blotting. The highest seropositivity was recorded among dogs from the eastern part of Slovakia, in the Presov (22.9%) and Kosice (17.1%) regions, long considered to be highly endemic for Trichinella occurrence and where the prevalence of infection in the vulpine population is also the highest in Slovakia. Trichinella-seropositive animals were detected significantly more often in dogs kept in rural areas (21.1%) when compared with animals living in towns or suburban localities (7.7%). On the other hand, neither age, nor gender and size of the dogs were shown to be a significant factor for Trichinella infection (P > 0.05). The present results indicate that the dog population in Slovakia is at high risk of exposure to Trichinella parasites and could potentially represent a suitable host group for serological monitoring of Trichinella infection as convenient sentinel animals.

Seroprevalence of human Toxocara infections in the Roma and non-Roma populations of Eastern Slovakia: a cross-sectional study.

Socioeconomic conditions and health of the Roma population, the most numerous minority in Europe, are worse than that of the non-Roma population. Information about the occurrence of human toxocarosis and other parasitic diseases in the Roma population is scarce or completely missing. The aim of this study was to map the seroprevalence of toxocarosis in the population living in segregated Roma settlements and to compare the data with the occurrence of antibodies in the non-Roma population of Eastern Slovakia. The seropositivity to Toxocara in 429 examined Roma inhabitants of segregated settlements reached 22·1%, while only 4/394 samples of the non-Roma population were found to be positive (odds ratio 27·7, P < 0·0001). Headache, muscle pain, influenza-like symptoms and diarrhoea occurred significantly more often in seropositive persons than in seronegative individuals. In the Roma population positivity was not influenced by gender, level of education and poverty, but age, lack of sanitary facilities and heating with wood significantly increased the risk of infection. It can be assumed that besides the high prevalence of toxocarosis, other parasitic diseases and communicable diseases will also be more prevalent in the Roma population living in segregated settlements.

First molecular detection of Neospora caninum in European brown bear (Ursus arctos).

Neospora caninum is an important intracellular protozoan parasite with an affinity to the central nervous system of many animals and a major causative agent of repeated abortions in cattle. In total, 45 muscle, liver, or spleen samples of brown bear sampled in different locations of Central Slovakia were examined by PCR. Genotyping of N. caninum was based on amplification of Nc-5 gene and ITS1 rRNA fragments and subsequent sequencing. Presence of N. caninum DNA was confirmed in 24.4% (11/45) of tested animals. The results obtained in study confirm the first molecular evidence of N. caninum DNA in European brown bear (Ursus arctos) in Slovakia.

Small mammals: paratenic hosts for species of Toxocara in eastern Slovakia.

Toxocara spp., an aetiological agent of a serious helminthozoonosis, is a common roundworm of domestic and wild carnivores worldwide. The study aimed to estimate the seroprevalence of Toxocara in small mammals from different localities in eastern Slovakia. Anti-Toxocara antibodies were detected in 6.4% out of 2140 examined animals trapped in eastern Slovakia. Due to their high density and observed high seroprevalence of toxocariasis, Apodemus agrarius, A. flavicollis, Myodes glareolus and Mus spicilegus (10.9, 4.2, 3.6 and 11.2%, respectively) represent important sources of the infection. A significant correlation between type of food and Toxocara positivity was detected: granivores (7.2%) and invertebratophages (7.1%) were positive more frequently than herbivores (2.1%). In the years monitored, cyclic changes of seroprevalence were observed. A higher prevalence of antibodies in the spring was followed by a decrease in summer. In autumn, seroprevalence started to rise and stayed at a similar level through the winter. Seroprevalence of the examined animals confirms their contact with Toxocara spp. and demonstrates the presence of the aetiological agent in the monitored locality. Areas with a high prevalence of infected animals present constant infectious pressure on definitive hosts, thus also increasing infection risk for humans and paratenic hosts. The study confirmed the contact of small mammals with Toxocara spp. and demonstrated the presence and circulation of an aetiological agent in the localities monitored in eastern Slovakia.

Influence of Echinococcus multilocularis infection on reproduction and cellular immune response of mice.

The influence of secondary Echinococcus multilocularis infection on reproduction and cellular immune response of mice was studied in BALB/c mice infected with 2000 E. multilocularis protoscoleces. Of the total infected mothers, 11.7% did not give birth and 10% of uninfected ones did not deliver. Both, healthy and infected mothers, produced on average 6-7 offspring per litter. The changes in production of seral IFN-gamma, TNF and IL-10 did not significantly influence the course of gravidity. On the other hand, more intensive metacestode growth was observed after the delivery. This study confirmed the ability of host organism to adapt to severe damage caused by E. multilocularis, not only in normal conditions, but also during pregnancy.

The first finding of Neospora caninum and the occurrence of other abortifacient agents in sheep in Slovakia.

Neosporosis is an infection of animals caused by an intracellular coccidian parasite, Neospora caninum, closely related to Toxoplasma gondii. The parasite is one of important abortifacient agents of bovine abortions worldwide. The aim of the study was to detect the prevalence of anti-Neospora antibodies in dairy aborting sheep from two eastern Slovak regions and to compare it with the occurrence of other potential abortifacient agents. Sera of 382 sheep, mainly the Improved Valachian and Merino breed, were tested for the presence of anti-Neospora and anti-Toxoplasma antibodies by ELISA, anti-Leptospira sp. by micro-agglutination-assay and anti-Chlamydophila antibodies using the complement fixation test. The mean seroprevalence of N. caninum was 3.7% and of T. gondii, 24.3%. This phenomenon of higher susceptibility of sheep to T. gondii is in the opposite of N. caninum infection in cattle. Anti-Leptospira antibodies were observed in 2.9% of serum samples with titres from 800 to 1600, whereas IgG antibodies against Chlamydophila abortus were found in 13.6% with titres from 64 to 1024. Half of N. caninum positive sera were simultaneously positive for T. gondii and one sample for C. abortus. From examined abortifacient agents the most important, from the frequency point of view, were toxoplasmosis (24.3%) and chlamydiosis (13.6%). No significant association between the frequencies of the abortions and mean seroprevalence of the abortifacient agents in Kosice region was determined. Likewise, no significant differences between the mean seroprevalence of neosporosis and toxoplasmosis in the two regions were detected. The first survey of neosporosis in aborting sheep from eastern Slovakia revealed a low prevalence resulting in a lower impact on reproduction losses in these small ruminants.

The first finding of Echinococcus multilocularis in dogs in Slovakia: an emerging risk for spreading of infection.

Alveolar echinococcosis, caused by the metacestode Echinococcus multilocularis, is a zoonosis of public health significance. Apart from red foxes, other carnivore species can also serve as definitive hosts of this parasite. This study aimed to determine the occurrence of E. multilocularis in dogs in the Slovak Republic and identify risk factors for acquiring the infection. Out of 289 dogs examined, E. multilocularis was detected in eight samples (2.8%). Positive results were observed in shepherd dogs (12.5%), guard dogs (7.1%), hunting dogs (2.4%) and in one dog with unknown usage (2.5%). Catching rodents and feeding with raw viscera were confirmed to be the most important risk factors for E. multilocularis infection. On the contrary, the way of dog use, frequency of going to the rural areas, the age and gender of animals seem not to be risk factors for infection. No significant association was observed between the positivity of dogs coming from high endemic or non-endemic regions. This is the first report of E. multilocularis in dogs in the Slovak Republic that confirms substantial contribution of dogs to the transmission of the tapeworm in this territory and urges the necessity of prophylactic measures for dog owners.

Humoral immune response of mice infected with low doses of Trichinella spiralis muscle larvae.

Serological techniques are frequently used to detect parasite status and to monitor epidemiology and disease prevalence in important reservoir hosts of zoonotic diseases. Small mammals present the most important link in the epidemiological chain in the spread of trichinellosis. In experimental studies, high infective doses are used to provoke strong immune response of laboratory animals. Wild animals, however, could be infected with very low numbers of Trichinella larvae. The aim of this work was to reveal the size of infective doses that can evoke an adequate immune response with detectable level of specific antibodies in mice. Sixty inbred (Balb/c) mice were infected with 50 L1 and 60 outbred (ICR) mice were infected with 5 L1 T. spiralis. The total larval burdens (TLB) in the intestinal and muscle phases, reproductive capacity index (RCI), and the kinetics of development of specific antibodies by iELISA with different conjugates were determined. In the first 10 days post infection (dpi), more adults were found in the intestines of inbred mice. In both mice strains, the first muscle larvae were observed at 20 dpi. The RCI was significantly higher in outbred mice. Sero-conversion of IgM antibodies was detected at 30 dpi. The IgG antibodies appeared at 40 dpi in inbred mice, and at 50 dpi in outbred mice. Using a polyvalent conjugate, the earliest sero-conversion was recorded at 30 dpi. Antibody levels increased until the end of the experiment (80 dpi). Our results support the suitability of ELISA in large epidemiological surveys to detect low-level infection in naturally infected small mammals, and are useful in epidemiological studies of the sylvatic circulation of trichinellosis to determine likely modes of transmission.

Neospora caninum, potential cause of abortions in dairy cows: the current serological follow-up in Slovakia.

Neosporosis is considered to be a contributing risk factor for abortions in dairy cows and other farm animals and has negative economic impact on their breeding. In respect of the rapid spread of neosporosis in herds throughout the world, our aim was to detect the prevalence of anti-Neospora antibodies in cows post-abortion (PA) (PA Group n=716) and in cows without any reproduction problems (Control Group n=247) on large breeding farms from Eastern Slovakia. The overall mean seropositivity in PA Group (20.1%) was significantly higher (p<0.0001) in comparison with the Control Group (2.3%), and this demonstrates the causal dependency of abortions on neosporosis. On farms from south-east region, 15.6% of cows were seropositive on average ranging from 7.8% to 25.8% in different districts. In the northern region, significantly (p=0.0002) higher 26.2% mean seropositivity was detected. The seroprevalence in different districts ranged from 2.9% to 39.4%. In the PA Group a high seropositivity to infectious bovine rhinotracheitis (41.5%) and sporadic occurrence of Q-fever, leptospirosis and chlamydiosis was also detected. From 11 Toxoplasma-seropositive cows, only one animal was simultaneously positive to Neospora. The relatively high prevalence rate in cows warrants the attention and a need of surveillance in Slovak herds. A better knowledge of epidemiology of this etiological agent and the mechanisms of its transmission may help in the introduction of more effective preventive and control measures.

Trichinella spiralis-outbreak in the Slovak Republic.

BACKGROUND: Trichinellosis is a zoonosis caused by nematode worms of the genus Trichinella and acquired through the consumption of raw or undercooked meat or meat products that harbour parasite larvae. The present report was designed to evaluate the course and circumstances surrounding the trichinellosis outbreak that occurred due to consumption of pork from a backyard pig in the southwest area of Slovakia in 2001. PATIENTS AND METHODS: Patients suspected of acquiring infection were serologically examined by ELISA, sera of patients with symptoms of clinical trichinellosis were additionally tested by Western Blot. In symptomatic patients haematological and biochemical parameters were assessed on day 45 p.i. RESULTS: An epidemiological investigation showed that the disease was linked to the consumption of infected pork and/or smoked pork products and affected the household members of four families. Out of 23 persons who had consumed the infected meat, 11 showed anti-Trichinella IgA, IgM and IgG antibodies in their sera by an ELISA against somatic and excretory-secretory antigens. Six of them developed clinical symptoms. Sera from symptomatic patients recognised bands of 64, 47, 45 and 43 kDa of crude T. spiralis antigens by Western blot. Using multiplex PCR analysis, parasite larvae isolated from pork were identified as T. spiralis, rarely occurring in Slovakia. CONCLUSION: The outbreak of human trichinellosis in an area where feral animals have been previously considered free of Trichinella was unexpected. Following the suspicion of trichinellosis being addressed, larvae were detected in meat and meat products from pig and the course of disease in patients was successfully controlled and managed.

Echinococcus multilocularis in the red fox Vulpes vulpes from the East Carpathian region of Poland and the Slovak Republic.

The occurrence of Echinococcus multilocularis in the Poland-Slovak frontier zone of the East Carpathian region was assessed, for comparison with that in adjacent regions in both countries. A total of 392 red foxes from Poland and 427 red foxes from the Slovak Republic were examined from 2001 to 2004. Significant differences in prevalences were observed in foxes captured from the borderland and adjacent zones in both countries. The mean prevalence of E. multilocularis in the Polish borderland reached 45.7+/-18.6% and in the Slovak border 35.0+/-10.7%. In both countries, the prevalence of E. multilocularis in red foxes from adjacent districts, outside the frontier Carpathian region, was considerably lower (18.9+/-9.2% in Poland and 20.8+/-9.0% in Slovakia). These differences are probably due to geomorphological and ecological factors, which contribute to the survival of the tapeworm eggs and the subsequent spread of infection. The Carpathian regions of northeast Slovakia and southeast Poland are characterized by specific climatic conditions such as low mean annual air temperatures, low temperatures in active soil surfaces, high soil humidities and a high mean annual rainfall.

Diagnosis and manifestation of encephalitozoonosis in mice after experimental infection with different species and application of dexamethasone.

Twenty-eight BALB/c mice were infected with different strains of Encephalitozoon species (Encephalitozoon cuniculi II - mouse type, E. cuniculi III - dog type, Encephalitozoon hellem, Encephalitozoon intestinalis). Five of them were infected with E. cuniculi II (mouse type) and simultaneously immunosuppressed with dexamethasone. Clinical signs of encephalitozoonosis were not remarkable. Ascites was found in two mice of dexamethasone-treated group 14 days post-infection (p.i.). The histopathological changes were found mainly in spleen and liver in the form of lymphoepithelioid granuloma. Spores were found in faeces since day 14 p.i. and visualized by Calcoflour White M2R. After cultivation on cellular cultures (VERO E6 - monkey kidney cells, RK-13 - rabbit kidney fibroblasts), the species differentiation was performed by PCR using panmicrosporidial primers (PMP1, PMP2) and specific primers (ECUN-F, ECUN-R, V1, SI-500). The differences were recorded in the immune response of immunocompetent and immunosuppressed mice. At day 60 p.i., the titres of specific antibodies measured by indirect immunofluorescence antibody test were lower (1:4096) in dexamethasone-treated mice when compared with non-immunosuppressed animals (1:8196). The significant increases of antibody titres were recorded in particular infected groups within the experiment (P < 0.01 between day 14 p.i. and day 30 p.i., P < 0.001 between day 14 p.i. and day 60 p.i.). Experimental encephalitozoonosis in non-immunosuppressed and immunosuppressed mice provides a useful model for the study of immune response and lesions associated with these protozoans.

Influence of Toxocara canis infection during pregnancy on offspring resistance towards re-infection.

The impact of Toxocara canis infection of Balb/c mice mothers on the future immune response of their offspring towards reinfection with the same parasite was studied. Two groups of offspring, the first originating from the mothers infected with a single dose of 1000 Toxocara canis eggs and the second from non-infected mothers, were both challenged with 500 T. canis eggs per animal at 6 weeks of age. The proportions of spleen CD4+ and CD8+ T lymphocytes, the level of serum cytokines IFN-gamma and IL-5, eosinophilia in peripheral blood, the production of specific antibodies and the number of migrating larvae were monitored. In both groups of offspring, the challenge infection resulted in an increase in CD4+ T-cell subtype in comparison with the non-infected healthy control, although after an initial decline a subsequent increase in CD8+ was observed. The immunoregulation index (CD4+/CD8+) was lower in the group of mice originating from infected mothers throughout the whole experiment compared to the offspring of non-infected mothers as well as in healthy control mice of the same age. In the offspring of infected mothers mainly, a reduced production of IFN-gamma and of IL-5, suppressed eosinophilia and a higher level of protective antibodies was detected, compared to the control second group, in which the INF-gamma concentration significantly increased after day 42 p.i. In the first group of offspring before challenge, 12.7 +/- 2.5 larvae in the brains and 32 +/- 2.1 larvae in the muscles transmitted from the infected mothers were detected. There was a significant reduction in larval recovery from brain on days 42 and 49 p.i. (56.7 and 56.8%, respectively), while from muscles in the same time there was a reduction of 46.7 and 39%, respectively, compared to the offspring of non-infected mothers. These results indicate a significant protective memory of immune mechanisms against T. canis induced in offspring of Toxocara-infected mother mice.

Field evaluation of an intravital diagnostic test of Echinococcus multilocularis infection in red foxes.

Echinococcus multilocularis parasitizes the small intestine of red foxes (Vulpes vulpes) and other carnivores, and has a wide distribution throughout the northern hemisphere. This cestode is the causative agent of human alveolar echinococcosis, a life-threatening helminth zoonosis. In 2000-2002, 2130 red foxes were examined for its presence in Slovakia, with a total prevalence of 30.7%. The data on occurrence were obtained by the combination of necropsy of small intestines from red foxes and coproantigen detection in faecal samples. The correlation between the number of detected specimens and the value of optical density of copro-ELISA test was found. When worm burdens were low (1-25 specimens) the sensitivity of the method was 31.3+/-8.64%, when worm burdens were >50 specimens, 81.8+/-0.66%, and with high worm burdens (>1000 specimens) the sensitivity reached 100+/-0.34%. E. multilocularis presence was detected using the nested PCR method from the eggs in the faecal samples with a 100% specificity. In epidemiological surveys of this zoonosis, it is of crucial importance to detect animals with a high level of infection, which are responsible for the bulk of environmental contamination. The advantage of copro-ELISA test lies in allowing the intravital diagnostics to be employed within the epidemiological survey of E. multilocularis occurrence in the protected and urban areas.

First record of Trichinella pseudospiralis in the Slovak Republic found in domestic focus.

Infection of Trichinella spp. is widespread among wildlife in Slovakia and the red fox (Vulpes vulpes) is the main reservoir of Trichinella britovi. Trichinella spiralis has been rarely documented in sylvatic and domestic animals of this country. During routine examination of domestic pigs at the slaughter, Trichinella larvae were detected by artificial digestion in a domestic pig of a large-scale breeding farm in Eastern Slovakia. The parasite has been identified by molecular (PCR) and biochemical (allozymes) analyses and by the morphology of the nurse cell as the non-encapsulated species Trichinella pseudospiralis infecting both mammals and birds. The epidemiological investigation carried out at the farm level revealed the presence of the same parasite species in other three pigs of 192 examined (2.1%), in 3 of 14 (21.4%) examined synanthropic rats (Rattus norvegicus) and in a domestic cat. The farm was characterized by inadequate sanitary conditions, insufficient nutrition, cannibalism and the presence of rat population. A different profile has been observed at the phosphoglucomutase locus in T. pseudospiralis isolates from Slovakia in comparison with the T. pseudospiralis reference isolate from the Palearctic region. This is the first documented focus of T. pseudospiralis from Central Europe. The detection in domestic pigs of a non-encapsulated parasite infecting both mammals and birds stresses the need to avoid the use of trichinelloscopy to detect this infection at the slaughterhouse.

Circulation of Toxocara spp. in suburban and rural ecosystems in the Slovak Republic.

Toxocara spp., the common roundworms of domestic and wild animals, are the causative agents of larval toxocarosis in humans. The migration of Toxocara larvae in men causes clinical syndrome, called larva migrans visceralis or larva migrans ocularis. The objective of the present work was to investigate the prevalence of toxocarosis in dogs, red foxes (Vulpes vulpes), and small mammals in the Slovak Republic. T. canis infection was diagnosed in 16.6% out of 145 dogs examined. Young animals showed significantly higher positivity (50.0%) than adults (12.4%). Coprological investigation of 310 red foxes showed 8.1% prevalence of T. canis, with higher occurrence in animals from rural conditions (8.6%) than from suburban environment (5.6%). Out of 710 small mammals examined, anti-Toxocara antibodies were detected in 7.7% of the animals. The most frequently seropositive species was Apodemus agrarius (15.9%). Seropositivity of small mammals from suburban localities was higher (10%) compared with rural areas (5.8%), with adult animals exhibiting higher seropositivity (8.0%) than subadults (6.8%). Our results have confirmed the importance of dogs, red foxes and small mammals in circulation of this serious helminthozoonosis in suburban and rural ecosystems.

Influence of maternal infection on offspring immune response in murine larval toxocariasis.

The impact of Toxocara canis infection on the proportion of CD4+ and CD8+ T splenocytes, the serum concentrations of cytokine IFN-gamma and IL-5, and the production of Toxocara-specific antibodies were studied in two C57BL6/J mouse groups and their offspring. The mice were infected with 1000 T. canis eggs on the day of mating (early infection) and on day 14 of pregnancy (late infection). Early infection resulted in a significant increase of CD4+ T-cell subtype, however, a decline in CD8+ in comparison with late infection, as well as with non-infected control. The IFN-gamma serum concentrations decreased in infected mothers after the birth when compared with non-infected mothers, while in the offspring this cytokine was barely or not detectable. In the mothers of both infected groups, the humoral immune response included both parasite-specific IgM and IgG2 antibodies. While IgG1 levels remained constant throughout the whole experiment in mothers with early infection, late-infected mothers became seropositive only 3 weeks after delivery. IgM was not detectable in any offspring. Pups from early-infected mothers had IgG1 antibodies. Conversely, IgG2 was detectable in pups of both experimental infection groups. A significant difference was observed in the amounts of pups/litter of the infected mothers in comparison with the non-infected ones. Only 56% of females after early infection and 79% of those after late infection had a successful pregnancy. However, all mice of the control group produced a litter. The first T. canis larvae were detected in the muscles of the offspring of both groups on day 5 after the birth. These data show the changes in regulatory and cytotoxic immunity mechanisms of the infected mothers and their offspring and the high level of pregnancy loss as a result of larval toxocariasis.

Trichinellosis in the Slovak Republic.

The occurrence of trichinellosis in farm or wildlife animals in some areas pose the permanent risk of infection in humans. In Slovakia, where only the sylvatic trichinellosis exists, the humans trichinellosis is sporadic. The most common cause of infection is wild boar meat. Unusual local eating customs may also favour an outbreak of disease in humans. The outbreak of trichinellosis in Slovakia in 1998, when 336 people were affected, was of the same kind. Trichinella britovi was the agent of the infection.

Congenital trichinellosis? Case report.

A large trichinellosis outbreak in the Slovak Republic caused by the species Trichinella britovi and affecting 336 people also affected a pregnant woman. The mother was infected in the 10th week of pregnancy and was treated with mebendazole. On her own request abortion was performed in the 22nd week of pregnancy. Medium IgM and high IgG anti-Trichinella antibody titres were found. The placenta, body cavities liquid, tissues and organs of the foetus contained 0.02-30 larvae per gram of tissue, measuring 0.68 +/- 0.05-1.17 +/- 0.07 mm, with blurred inner structure. Immunocytochemical examination identified Trichinella larvae that infected the foetus in the early stage of development.