Presence of gastrointestinal parasites in swine and human of four swine production farms in Cundinamarca-Colombia

Objectives. Determine the presence and the type of endoparasites with zoonotic potential in swine and human of two technified and two semi-technified farms in the department of Cundinamarca, Colombia. Materials and methods. Three serial samplings of feces were taken in a pen row within intervals of 15 days, in two technified and two semi-technified farms in different age groups distributed as follows: pregnant-sows, nursing-females, boars, weaners, suckling-piglets, and growing-pig. By means of informed consent thirty-three people agreed to enter the study. Thirty-three samples from men and women of different ages were received. The pool and individual samples of fecal were evaluated by direct analysis, qualitative flotation and sedimentation techniques and modified Ziehl-Neelsen stain. Results. For the porcine population, on the average, the results obtained from both technified farms showed that Balantidium coli (42%), Endolimax nana (21.9%) and Iodamoeba bütschlii (7.8%) were the most common parasites. In semi-technified farms they were: Entamoeba coli (40%), Endolimax nana (35%), Iodamoeba bütschlii (25%) and Balantidium coli (5%). By means of the test chi 2 it is possible to conclude that there is a significant difference between the parasites species and the type of farm. The results obtained in human showed the presence of parasites as: E. coli (42.2%), Entamoeba hystolitica/dispar (12.1%), E. nana (9.1%), B. coli (9.1%), I. bütschlii (3.0%) and Blastocystis hominis (3.0%). Conclusions. The presence of parasites such as Balantidium coli, Endolimax nana, Iodamoeba bütschlii and Entamoeba coli in swine and human suggests a possible rotation of parasitic species between hosts.


INTRODUCTION
In Colombia, during the year 2013 the porcine production was 670.612 animals, to which Cundinamarca contributed 4.98% (1); 1.458.179pigs were slaughtered for human consumption between January and June of 2013, Cundinamarca contributed 20.1% (650.035pigs) (1).This type of animal production is affected by the presence of parasites that limit the productive potential of the animals, thus affecting the average daily weight gain, causing heterogeneous lots as well as facilitating weight decrease during weaning, which seems reflected in the alteration of the food conversion indexes and further represents an increase in the time required before slaughter (2).It is important to note the rate of prevalence and the economic importance of the parasites changes depending on the management system, the housing characteristics, the hygienic-sanitary measures, the geographic location and the age of the pig.Thus, all these variables influence the basic requirements of every stage of the parasitic cycle, in both the mechanisms of transmission and the immune response of the host (3).
The parasites that have a major impact on health and porcine production are protozoan parasites such as Eimeria spp., Isospora spp.and Balantidium spp.; worm infections reported were primarily nematodes (such as Trichinella spp., Strongylus spp., Ascaris spp., Trichuris spp., Oesophagostomum spp.and Metastrongylus spp.) secondarily trematodes (such as Fasciola spp.and Dicrocoelium spp.) and finally cestodes (such as Echinococcus spp.and Taenia solium) (4,5).Many of these pathogens affect porcine production and can be transmitted to humans, and are recognized as zoonotic agents (6) among these we can find Balantidium coli, Toxoplasma gondii, Cryptosporidium spp.and Taenia solium (7).
Los parásitos que tienen mayor impacto sobre la salud y la producción porcícola son protozoos como Eimeria spp., Isospora spp.y Balantidium spp.; en cuanto a los helmintos se reportan principalmente nemátodos (Trichinella spp., Strongylus spp., Ascaris spp., Trichuris spp., Oesophagostomum spp., y Metastrongylus spp.), en segundo lugar tremátodos (Fasciola spp., y Dicrocoelium spp.) y finalmente céstodos (Echinococcus spp., y Taenia solium) (4,5).Muchos de los patógenos que afectan la producción porcina pueden ser transmitidos al ser humano, por lo que son reconocidos como agentes zoonóticos (6) entre los que se In Colombia, few epidemiological studies have been made in the matter, which is why not much information is known about the prevalence of porcine parasites.Therefore, it is of vital importance to conduct studies on the topic in order to know which are really parasites of zoonotic importance in this country.Thus the goal of this study was to determine the presence of gastrointestinal parasites (GIP) in swine and human who could be involved with zoonoses at four swine farms in the department of Cundinamarca -Colombia.

MATERIALS AND METHODS
Farm's Location.Two technified swine farms (T) (≥ 200 females) and two semi-technified farms (ST) (≤ 200 females) with a complete cycle of production with an all in/all out system (AI-AO), were chosen in the following locations

Sample size.
Bearing in mind that the total population was less than a 100.000(finite population), the following formula was used to calculate the sampling size (Table 1).

Sample collection.
Collective and individual samples (Figure 1) were obtained according to the phases of the productive cycle as follows: Group 1 Individual Samples: Pregnant sows, Nursing Females, boars.
Three samples were taken within an interval of 15 days each.
The farm workers and some of their relatives gave us a stool sample;

Sample processing
Macroscopic and microscopic approach.Macroscopic aspects such as color, consistency, presence of mucus, blood and/or parasites/ fragments were evaluated.
A direct examination was conducted in saline and lugol solution, under 10x and 40x magnification; the report of the results obtained was made using a semi quantitative scale with crosses, as follows: One cross (+) if 1 to 3 parasites per field are present.Two crosses (++) if 4 to 7 parasites per field are present.Three crosses (+++) if 8 to 10 parasites per field are present.Four crosses (++++) if more than 10 parasites per field are present.(13,14) Semi quantitative concentration method based on flotation.2 g of fecal matter were deposited in a wide flask with a saturated solution of sodium chloride and glucose, the procedure was made according to what is established in the guide RVC/FAO (15).A direct examination was conducted in the microscope, under 10x and 40x magnification; the report of the results obtained was made using a semi quantitative scale with crosses as previous described (13,14).
Semi quantitative concentration method based on sedimentation. 2 g of fecal matter were homogenized in water; the procedure was made according to what is established in the guide RVC/ FAO (15), finally with the aid of a pipette Pasteur the sediment was taken and a drop of it was placed in a microscope slide, using magnification 10x and 40x; the report of the results obtained was made using a semi quantitative scale (13,14).
Ziehl-Neelsen modified method.From the sediment taken, a smear study was made on a slide as follows: first by adding carbol fuchsin for 5 min and then washed with abundant water, later acid alcohol was added up to the complete discoloration for not more than 3 minutes, then Todas las personas que participaron en el estudio firmaron un consentimiento informado según lo establecido en el Decreto 3380 de 1981, artículo 10 y la Ley 23 de 1981, en donde se establecen las leyes de ética médica.

Statistical analysis.
To determine the relationship between the parasites species Vs. the production system and the age groups, the statistical test Chi 2 was applied, additionally the portion of parasites was determined by the test of proportion hypothesis.The data analysis obtained in the human samples was done by the presence or absence of parasites.

RESULTS
Porcine population.In the farm T1 a high presence of Balantidium coli 36.6% was found, followed by Ascaris suum 29.2%, Endolimax nana 15.2%, Giardia spp.12.1% and by a low prevalence of Iodamoeba bütschlii 7.5% and Trichuris suis 4.8%.On the other hand in farm T2 a high presence of B. coli 48.5% and E. nana 26.4% was found, followed by a low presence of I. bütschlii 8.2% and Strongyloides spp.5.8%.(Table 2) Fort the semi-technified farms, in ST1 a high presence of I. bütschlii 57.1%, B. coli 33.3%, E. nana 14.2% was found, followed by a low presence of Cryptosporidium spp.5% and E. coli 4.7%.Finally in farm ST2 the parasites with a high presence were: E. coli 40%, E. nana 35%, I. bütschlii 25%, Isospora spp.25%, Eimeria spp.20% and a low presence of B. coli 5%.(Table 3)  Análisis estadístico.Para determinar la relación existente entre especie parásita vs sistema de producción y grupos etarios se realizó la prueba estadística Chi 2 , adicionalmente se determinó la proporción de parásitos en las granjas tecnificadas y semi-tecnificadas, esto se realizó por medio de la prueba de hipótesis con proporciones.El análisis de los datos obtenidos en las muestras humanas, se realizó por presencia o ausencia de parásitos.In Colombia only a few epidemiological studies about this topic have been made, thus for this reason the data about the prevalence of swine parasites is yet unknown.The results obtained    (20), as it was shown in the current study where young pigs showed a higher presence of some parasites.(Table 2, Table 3).

RESULTADOS
According to the data obtained and analyzed by means of the statistical Chi 2 test, this study proves the existent relationship between the presence of parasites and the type of farm analyzed (semi-technified and technified farms) with a value of p<0.001 indicating a higher presence of parasites in the semi-technified farms; simultaneously it shows an existent relationship between the presence of parasites and the age group involved with a value of p<0.001.Additionally, when the test of proportion hypothesis was done it was able to determine that B. coli, was found in a higher proportion in technified farms than in semi-technfied farms with a value of p<0.001.E. nana and I. bütschlii were found in a higher proportion in the semi techn farms than techn ones with a value of p<0.01 and p<0.001 respectively.
It is important to acknowledge that the higher prevalence of parasites is found in the nursing females, pregnant sows, weaners and suckling piglets in technified as in semi-technified farms as well, this higher presence in these age groups is possibly given due to the physiological conditions in which the animals are living; in the females, the stress conditions and well as the immune suppression physiological process they deal during their pregnancy period, making them more susceptible to infections by diverse parasites, thus constituting the main source of infection for the weaners (4) additionally, studies have proved that the age of the weaners is a critical factor in parasite infections, due to the high sensibility of piglets to infections by their immune immaturity in their first Es importante reconocer que la mayor prevalencia de parásitos se detectó en las hembras lactantes, las hembras gestantes y en los lechones lactantes y destetados, tanto en las granjas tecnificadas como en las semi-tecnificadas; esta mayor presencia en los grupos de edad se debe, posiblemente, a las condiciones fisiológicas en las que viven los animales; en las hembras, las condiciones de estrés así como el proceso fisiológico de inmunosupresión que afrontan durante su período de preñez, las hacen más susceptibles a las infecciones por diversos parásitos; constituyendose estas en la principal fuente de infección de los lechones destetados (4); adicionalmente, estudios han demostrado que la edad de los lechones destetados es un factor crítico en las infecciones parasitarias three days of life, whereas they achieve a high immunity against infections by their 2 week, (4).
As a opposing argument to the report of the current study in, the suckling piglets with an average of 5 weeks of life, were found with a higher prevalence of parasites in both types of farms, a 50% in technified farms and a 81.3% in semi-technified farms.
It has been proven that the prevalence of parasites varies depending on the management, location, weather and deworming processes implemented in each one of the farms.( 21) One of the reasons why it is believed that the presence of parasites varies from one farm to another is the weather conditions; this data is relevant for the association of certain parasite species, as the samples in both technified farms were done during the months of August and September in 2012, and according to the report by the hydrology, meteorology and environmental studies institute (IDEAM) ( 9), the Cundinamarca zone showed pluviosity excess (8).This could be related to the absence of some parasites that are reported in literature and were not found in the current study, such as, Oesophagostomum spp., that reports an optimum temperature for its development of 25°C, (10) whereas for Eimeria spp.and Cryptosporidium spp. a temperature between 21 y 22°C is reported and optimal dry conditions for its development and latter infection (21).Thus, the above might be one of the reasons by which these parasites were not found in farms T1 and T2, due to the temperatures and pluviosity in both locations during the months of the study, and so not helping the infection of these parasites.
On the contrary, in the semi-technified farms the presence of Eimeria spp., Isospora spp.and Cryptosporidium spp.were evident and the presence Oesophagostomum spp. was not found, thus this might be related to the weather conditions during the months of April and May of 2013, during which the samples were taken and had a dry weather as reported by IDEAM.( 9) Another important condition related to the prevalence of parasites are the hygienic and sanitary conditions managed by each farm, as it can be clearly seen in the semi-technified farm ST2, were the water used for animal consumption came from a natural source without any type of treatment due to the owner of the farm who considers that the water is completely pure and so it does not need any treatment whatsoever, and thus this might be related to the high prevalence and variety of parasites found there.
In the present study I. bütschlii was found, and it is reported in literature as an intestinal non pathogen amoeba in humans, apes and swine; even though it was described as the most common intestinal amoeba in swine (22) its presence has not been reported in this host; only one study made in twelve boar intestines in Iran during 2004 showed a prevalence of 17% of I. bütschlii (7).
It is of relevance to acknowledge, the presence of Cryptosporidium spp. in a semi-technified farm in a very low proportion (1+) in three different age groups; the infections by this agent have a higher frequency rate in early stages as weaners and suckling piglets, whereas in adults is slightly probable; the absence of this specie in the other farms might be due to the preventive measures taken such as a pretreatment with coccidiostatics in early stages.On the contrary to the report of the literature in the current study there was evidence of the presence of Cryptosporidium spp., in suckling piglets and females stages (23).
The consistency of the fecal matter samples analyzed from humans in its majority was very soft but not watery.According to the results found in the sampling procedure done to some of the workers of both technified and semi-technified farms, a possible zoonotic process might be correlated with B. coli were the pigs the reservoir of the parasite and the main source of infection for the human (24).The presence of this parasite in humans is conditioned to bad health conditions, lack of basic utilities (water, sinks) and the breeding of the swine in open fields (10), this last aspect was not prevalent in the current study, due to the breeding swine in barns; nevertheless, the conditions and sanitary-hygienic habits of the humans are possibly the highest risk factors in the zoonotic processes.
In Latin America, the prevalence of the infection by B. coli ranges between 0.5 and 2.1% (21), showing low among other protozoan intestinal infections.The quality of the water and thus the presence of parasites, to establish with certainty what is the true source of infection and the existence of transmission between species.A wide study done in Bolivia in 2.000 samples of feces coming from Aymara native children from the Bolivian steep lands region, showed a disseminated infection by B. coli with a general prevalence of 1.2%; besides this, swine fecal samples were analyzed, and the results showed that more than half of the swine (n=50) living in this native community were infected by B. coli, wich clearly indicates En el presente estudio se encontró I. bütschlii que es reportada como una ameba intestinal no patógena en los humanos, los monos y los cerdos; aunque fue descrita como la ameba intestinal más común de los cerdos no se ha reportado su presencia en este hospedero; solamente un estudio realizado en 12 intestinos de jabalíes examinados en Irán en el año 2004 se encontró una prevalencia del 17% para I. bütschlii (7).
En América Latina la prevalencia de la infección por B. coli en humanos, oscila entre el 0.5 y el 2.1% (21), siendo baja frente a otras infecciones intestinales por protozoos.Son necesarios estudios adicionales donde se evalué la calidad del agua y presencia de parásitos para saber con certeza cuál es la verdadera fuente de infección y si hay transmisión entre especies.Un amplio estudio realizado en Bolivia con 2.000 muestras de heces procedentes de niños Aymaras nativos de regiones escarpadas bolivianas, mostró una infección por B. coli con una prevalencia general de 1.2%; adicional a esto se analizaron muestras de heces de cerdos y los resultados mostraron que más de la mitad de los cerdos (n=50) que viven en esta a zoonotic transmission (5).The illness in swine is asymptomatic, whereas, in humans it might show clinical outcomes ranging from asymptomatic to severe ones, where the asymptomatic ones serve as reservoirs and disseminators of the illness; diarrhea, blood feces, cramps and abdomen pain is shown in the severe ones (5).
A study made in Egypt, showed the presence of de E. coli and E. histolytica/dispar in samples of swine and dogs in a 7.56% y 3.84% respectively, additionally samples of children with diarrhea were analyzed and showed a high prevalence of both species, thus suggesting a possible anthropozoonotic infection with dogs and swine in the area (25).Additionally, during the current study the presence of E. nana was evidenced, and from which its zoonotic potential has not been established yet, but it is said that it might be involved in zoonosis were swine and humans are in high contact with the infective form (Cysts) by water consumption, food or contaminated hands and thus helping the zoonotic pathogens transmission between species, causing disease (7,26).
A study made in the Republic of Korea, related to zoonotic parasites reported that the main parasites with zoonotic potential are E. nana and I. bütschlii, which are protozoans with sapro-zoonotic potential, meaning humans become infected by contaminated food or water with feces of swine, dogs and cattle (7).
Another parasite found was Blastocystis hominis, which pathogen potential in humans and swine is still controversial due to the absence of symptoms, the presence of this microorganism in feces of a variety of animal species such as birds, apes, rodents, amphibians and humans suggests the rotation among them, thus considered a protozoan with zoonotic potential (7,26), also it is associated with the consumption of water, fruits and vegetables contaminated with feces.This parasite is found in a high prevalence in human population, for example, in Argentina in a 43% of children in school age was found, it has been registered in Venezuela with a prevalence of 27%, from which 24% of children in school age, mainly between the age of 9 and 11 years old and 23.9% of the elderly population were infected by this protozoan.(23) Although in the analyzed humans, were not observed compatible structures with A. suum, T. suis and Cryptosporidium spp., it is of importance to stand out the zoonotic potential of these parasites.Ascaris lumbricoides and comunidad nativa estaban infectados por B. coli, lo cual claramente indica una transmisión zoonótica (5).La enfermedad en los cerdos es asintomática mientras en los seres humanos podría mostrar resultados clínicos que van desde asintomáticos hasta severos, donde los asintomáticos sirven como reservorios y diseminadores de la enfermedad mientras en los severos se pueden presentar diarrea, deposiciones con sangre, calambres y dolor abdominal (5).
Ascaris lumbricoides y Trichuris trichiura infectan a más de mil millones de personas en todo el mundo Trichuris trichiura, infect more than a thousand million people worldwide (27), the species A. suum and T. suis in swine have a global distribution, due to the presence of infected swine mainly in the production system (27).Swine and humans alike are infected by the ingestion of the eggs that acquire an infectious stage in the environment (26).This type of parasitosis produces high economic impact, due to the frequent delay in growth and diarrhea in swine.The differentiation between human and swine parasites species has been hard and due to this reason the frequency of infection of A. suum and T. suis in humans cannot be determined (26), but a zoonotic implication is not discarded due to it similarity and the possibility to infect humans especially in areas where swine and humans live close to each other or swine feces is used as agricultural fertilizer in vegetables for human consumption or by water pollution and bad hygiene of workers in the swine farms, thus existing a potential risk of crossed infections (27).
The cryptosporidiosis is a disease of high zoonotic importance, known worldwide as a public health problem that affects mainly people that interact with farm animals on a daily basis (5).Generally, the infections by Cryptosporidium in domestic swine are higly frequent in animals between 1 and 6 months old, reaching a prevalence between 24 and 60%; the 16.5% of the adults had an infection by Cryptosporidium spp.(21), this data supports the fact that adult swine are parasited in a lower degree (21).
Recent studies have showed the existence of Cryptosporidium suis, finding done by means of molecular biology which evidenced that this is genetically different from the other species of Cryptosporidium known (21).
It is relevant to evidence the presence of pathogens with zoonotic potential, thus they might prove to become relevant public health issues, besides, pitching in knowledge concerning these type of parasites, will make decision making in preventive measures that deny the rotation of parasites between animal and humans.
Infection due to contaminated water, bad human hygienic practices contributes to the transmission of parasites to swine, and thus these act as a reservoir disseminating the parasites by fecal means.When used as agricultural fertilizer it contaminates crops and water supplies acting as a source of infection for humans and animals alike in an endless cycle.
La infección por agua contaminada y las malas prácticas de higiene de los humanos contribuyen en la transmisión de parásitos a los cerdos y estos actúan como reservorio diseminando los parásitos por medio de las heces, las cuales al usarse como abono en agricultura contaminan las hortalizas y las aguas siendo fuente de infección tanto para los humanos como para otros animales siguiendo un ciclo sin fin.

Farm 1 (Farm 2 (Farm 3 (Farm 4 (
T1): Located at a height of 1.728 (m.a.s.l.), with an average temperature of 20°C (8) and a population of 3654 pigs.T2): Located at a height of 2.586 (m.a.s.l.), with an average temperature of 14°C (8) and a population of 2156 pigs.ST1): Located at a height of 1.867 (m.a.s.l.), with an average temperature of 20°C (9) and a population of 244 pigs.ST2): Located at a height of 1.384 (m.a.s.l.), with an average temperature of 20°C (9) and a population of 125 pigs.

Figure 1 .
Figure 1. A. Individual sample gathering -Manual stimulation of the rectum B. Collective Sample gathering.

Table 2 .
Presence of gastrointestinal parasites per age group in technified swine farms by percentage. B.

Table 3 .
Presence of gastrointestinal parasites per age group in semi-technified swine farms by percentage.
B. coli E. nana I.

bütschlii Isospora spp. Eimeria spp. Entamoeba coli Cryptosporidium spp.
study vs other studies related with to the presence of parasites such as B. coli, A. suum, T. suis, Giardia spp., Strongyloides spp., Eimeria spp.and Cryptosporidium spp., match it; whereas differs in the findings of Oesophagostomum spp., because the last one was not found in the present study.