First report of Nematodirus filicollis natural infection in a sheep from the mexican sub-humid tropics Primer reporte de infección natural de Nematodirus filicollis en un ovino del trópico sub-húmedo mexicano

Objective. The aim of the present study is to report for the first time the natural infection of a sheep with Nematodirus filicollis in the sub-humid tropics of Mexico. Materials and methods. Faecal samples were processed to identify and quantify faecal nematode eggs per gram of feces (EPG) using the flotation and modified McMaster techniques. Also, faecal cultures were made using the Corticelli-Lai technique. Results. The ewe showed a mixed infection with Trichuris spp. (100 EPG), Nematodirus (100 EPG) and other strongylida nematodes (1500 EPG). The egg size of Nematodirus spp. was 202±3 μ long and 97±2 μ wide. Larvae of Haemonchus, Trichostrongylus, Oesophagostomum and Nematodirus genera were identified from de faecal cultures. Nematodirus larvae averaged 955±8.0 μ long and 28±0.5 μ wide. Whereas, the average length of the sheath tail was 233.4±41.6 μ. Conclusions. According to the morphological traits of eggs and larvae, it is concluded that the infection of the sheep corresponded to Nematodirus filicollis. It is concluded that N. filicollis is able to survive in the sub-humid tropics of Mexico and for the first time an acquired natural infection of this nematode in a sheep is reported.


INTRODUCTION
The genus Nematodirus Ransom, 1907 corresponds to nematodes of the small intestine of ruminants that have a direct life cycle and are members of the subfamily Nematodirinae (Nematoda: Trichostrongyloidea).The genus Nematodirus is composed of more than 45 species and many are distributed in ruminants mainly from the United Kingdom, Norway, New Zealand, Canada and the United States of America (1, 2).
In the United Kingdom, N. battus produces the death of thousands of lambs annually.In sheep herds, morbidity and mortality can reach 50-100% and 5-20%, respectively, with an impact on the welfare and economy of the farms (3).
In a phylogenetic study conducted by Hoberg (4) he showed that the Nematodirinae subfamily originated in the Holarctic with primary distributions determined through the Beringia.The stages of development of free life have adapted to extreme weather conditions, being relatively resistant to low temperatures and drying (4).
The genus Nematodirus (eg, N. battus, N. filicollis) has been mainly restricted to temperate regions of the northern hemisphere (3,5).In order for the third stage larvae (L 3 ) to emerge, the embryoed egg must be exposed to low temperatures (close to 0 ° C) for a prolonged period, followed by an increase in temperature above 10°C (6).However, N. battus has been reported in Sicilia, Italy (7), under conditions where the minimum temperature in winter sporadically reaches <11°C in this Mediterranean island.This adaptation to various temperate climates serves to concentrate the infecting larvae in the spring when there are susceptible lambs, limiting the reproduction of this nematode to one generation per year (8).
In Mexico, the genus Nematodirus has only been reported in sheep from regions with subtropical and temperate climates (9)(10)(11), where climatic conditions are favorable for the development of infective larvae and allows for the infection of vulnerable sheep.So far the genus Nematodirus has not been reported as a natural parasite of sheep in tropical climates in Mexico, for this reason this study reports for the first time, the case of a sheep that acquired the natural infection with Nematodirus spp. in sub-humid tropical climate conditions.
Manejo del rebaño.Todos los ovinos de la granja son mantenidos en confinamiento y reciben alimento en corrales de tierra.Los

MATERIALS AND METHODS
Design.The report corresponds to a case study, with epidemiological circumstantial evidence related to its occurrence.In the Veterinary Parasitology laboratory of the Campus of Biological and Agricultural Sciences of the Universidad Autónoma de Yucatán (CCBA-UADY), the feces of a 24-month-old female sheep of the Katahdin breed were registered for the diagnosis of gastrointestinal parasites.The sample came from a sheep farm located in the municipality of Mérida in the state of Yucatán, Mexico.In the coproparasitoscopic examination the presence of eggs of Nematodirus spp.(Figure 1) was observed.Given this finding, we proceeded to visit the farm to carry out an epidemiological study and to obtain the possible origin of this nematode.Relative humidity varies from 65 to 90%, with an average of 80% and annual rainfall of 1,000 mm.Two annual seasons are present: rainy (from June to November) and dry season (from December to May) (12).The geomorphology belongs to the young carsotectonic system, classified as a geomorphological landscape of undulating structural plains with dissolution and denudation.Soils are mainly lithosol, rendzin and cambisol (13).

Clinical examination of sheep and herd.
The physical examination of the sheep was performed (Figure 2) and pale mucous membranes (FAMACHA ® of 3, score of 1-4), body condition of 3 (score 1-5) and diarrhea were found.The ovine positive to Nematodirus spp. was sampled again to confirm the infection along with 12 animals that remained in the same pen for the previous three months.Additionally, the three sheep from  14), the first portion immediately after harvesting was used to perform larval culture and the second was preserved for 21 days under refrigerated conditions (1-2°C) to later perform the culture of larvae in an incubator at 28°C.The larval L 3 harvests of both cultures were carried out after 21 days.The classification of larvae L 3 was determined by their morphology and size (8).In order to identify the species of the genus Nematodirus 100 larvae L 3 were measured (width, total length and length of the tail) with a micrometer adapted to an optical microscope (10 x).

Database of the Veterinary Parasitology
Laboratory of the CCBA-UADY.To know the existence of cases of Nematodirus spp. in ovines from the state of Yucatan, Mexico, the files of the Veterinary Parasitology Laboratory of the CCBA-UADY were reviewed from January 2008 to September 2016.Additional information was also obtained on positive cases such as the origin of the animals and the elimination of eggs per gram of feces.

RESULTS
Table 1  All eggs of Nematodirus observed in the ovine feces presented thin cover, without color and conical ends (Figure 3).The size of the eggs was 202 ± 3 μ long and 97 ± 2 μ wide.
The fact that the eggs are colorless, their morphology with conical ends and size ruled out the presence of Nematodirus battus as an infecting species.
The 12 sheep that remained in the pen with the positive ovine for Nematodirus spp.and the three sheep from the municipality of Córdoba, Veracruz did not eliminate eggs of Nematodirus spp.
Both the culture carried out without the previous cooling as well as the one that was carried out with one week cooling allowed the recovery of larvae of Nematodirus spp.The genera of nematodes identified in the larval cultures without the previous cooling of the faeces corresponded to Haemonchus (58.1%),Trichostrongylus (32.5%),Oesophagostomum (5.8%) and Nematodirus (3.4%).Likewise, the genera of nematodes identified in the larval cultures with the previous cooling of faeces corresponded to Haemonchus (59.0%)Nematodirus (24.4%),Trichostrongylus (15.1%) and Oesophagostomum (1.6%).
The larvae of Nematodirus spp.found in the sheep agree with the descriptions of other Las larvas de Nematodirus encontradas en el ovino concuerdan con las descripciones de otros autores por ser fácilmente reconocibles debido a su gran tamaño y longitud y la forma filamentosa como termina la cola de la vaina.
La obtención de larvas L 3 de N. filicollis a partir de los cultivos de laboratorio con y sin previo enfriamiento de la heces, permite suponer que este género no requiere obligatoriamente de un periodo de enfriamiento y por ende puede authors because they are easily recognizable due to their large size and length and the filamentous shape at the ends of the sheath tail.The average size of the larvae of the Nematodirus genus identified in the sheep was 955 ± 8.0 μ in length and 28 ± 0.5 μ in width.
While the average length of the sheath tail was 233.4 ± 41.6 μ.The anterior tip of the larvae is rounded, with an oral cavity in the form of a straight tube and a filariform esophagus.There are eight large intestinal cells, well delimited with their respective nucleus.In the hind limb, the tail of the larvae have a fish tail shape that characterizes N. filicollis.

DISCUSSION
Several authors mention that for a natural infection of the Nematodirus genus to exist in sheep, most species require eggs to be exposed for prolonged periods at low temperatures (close to 0°C), followed by an increase in temperature higher than 10°C (6).However, Torina et al (7) report the natural infection of sheep with N. battus in Sicilia, Italy, where the minimum temperature in winter sporadically reaches <11°C.This last situation could have occurred in the farm studied since the natural infection occurred in a sub-humid tropical climate where temperatures of less than 10°C can only be recorded in January and February (12).In an experiment conducted by Van Dijk and Morgan (15), they compared the hatching requirements between N. battus isolates from Scotland and southwestern England and found that not all larval eggs require cold stimulation to produce infective stages.Likewise, Van Dijk and Morgan (3) mention that prolonged periods of high temperatures (25-30°C) do not necessarily affect the survival of N. battus eggs.It would be expected that high temperatures increase the mortality rates of infective larvae, but management practices such as the increase in animal load favors the infection of sheep with this nematode and allows its survival in extreme climates (3,16).
Obtaining larvae L 3 of N. filicollis from laboratory cultures with and without previous cooling of the feces, allows us to suppose that this genus does not necessarily require a cooling period and therefore can survive in the environmental conditions of the subhumid tropic.Likewise, the establishment of the genus Nematodirus in other regions that are not temperate such as in Sicily, Italy (7), Hueytamalco, Puebla, Mexico (10)  La baja eliminación de huevos (100 h/gh) de N. filicollis en el ovino del presente estudio y las limitantes climáticas del trópico mexicano para la sobrevivencia de los huevos larvados del nematodo podrían explicar la baja transmisión en el rebaño estudiado.Además, el sistema de producción intensivo que se manejó en la granja de estudio y la fertilización del pasto de corte con heces de animales de la misma granja, incluyendo las heces de los animales que fueron introducidos de Córdoba, Veracruz (animales sospechosos de introducir el género de nematodo en la granja), pudieran explicar la transmisión de este nematodo en el trópico mexicano.En futuros estudios será necesario evaluar la capacidad de N. filicollis para producir infecciones naturales en distintos sistemas de producción y manejo de ovinos criados en el trópico mexicano.
of areas where the eggs are cooled to produce infective larvae.
In a study conducted by Sánchez and Quiroz (11) 2% and 1% of sheep from Tlaxco, Tlaxcala, Mexico (cold weather 2603 m above sea level) were parasitized by N. battus and N. spathiger respectively.Most studies in Mexico report the presence of the Nematodirus genus without identifying the species of this nematode (9,10).The present study is the first report for Mexico of N. filicollis in tropical climate; although it was not possible to obtain the adult parasites of N. filicollis because the infection occurred in a female registered ovine and the owner did not allow the sacrifice of the animal.In future studies, it will be necessary to obtain the adult stages and compare them morphometrically and genetically with other Nematodirus species and evaluate the nematode's capacity to cause damage to the health of the sheep of the Mexican sub-humid tropics.
The low elimination of eggs (100 EPG) of N. filicollis in the ovine of this study and the climatic limitations of the Mexican tropics for the survival of the larval eggs of the nematode could explain the low transmission in the herd studied.In addition, the intensive production system that was handled in the study farm and the fertilization of the cutting grass with feces of animals of the same farm, including the feces of the animals that were introduced from Córdoba, Veracruz (animals suspected of introducing the nematode genus into the farm), could explain the transmission of this nematode in the Mexican tropics.In future studies it will be necessary to evaluate the capacity of N. filicollis to produce natural infections in different production and management systems for sheep raised in the Mexican tropics.
The mixed infection of N. filicollis with other nematodes of the strongylida order along with the genus Trichuris found in the study sheep could have contributed to the deterioration of the health of the animal and present a clinical picture of gastrointestinal nematodiosis, a condition frequently observed in sheep of the region (17).
In conclusion N. filicollis is capable of surviving t he b i o g eo g rap hi c al and c l i mat o l og i c al conditions of the state of Yucatan, Mexico, and for the first time the case of a sheep that acquired the natural infection with N. filicollis in tropical sub-humid conditions is reported in Mexico.
Se concluye que N. filicollis es capaz de sobrevivir a las condiciones biogeográficas y climatológicas del estado de Yucatán, México y se reporta por primera vez el caso de un ovino que adquirió la infección natural con N. filicollis en condiciones de clima tropical sub-húmedo en México.

Agradecimiento
Se agradece al propietario de la granja ovina por permitir el acceso a su predio y realizar el estudio.

Figure 1 .
Figure 1.Nematode egg of the order strongylida (A) and egg of Nematodirus spp.diagnosed by the centrifuged flotation test in a sheep born in the sub-humid tropics of Mexico (the bar corresponds to 10 μ in length).

Figure 2 .
Figure 2. Female sheep positive for Nematodirus spp.in the sub-humid tropics of Mexico.

Figure 3 .
Figure 3. a. Larva of Nematodirus spp.recovered from the Corticelli-Lai larvae culture of a sheep born in the sub-humid tropics of Mexico (bar corresponds to 100 μ in length).b.End of larva tail unsheathed from Nematodirus filicollis.
Rodríguez-Vivas et al -Nematodirus filicollis en ovino del trópico sub-húmedo Herd management.All the sheep in the farm are kept in confinement and receive food in earthen pens.Juvenile animals are raised in high cages with a slat floor.The feeding consists in the administration of foliage of grasses (Pennisetum purpureum, Penissetum violaceum) and arboreal (Brosimum allicastrum, Guazuma ulmifolia and Leucaena leucocephala) grown in the same farm.The grass is fertilized with feces from the sheep in the farm.Additionally, sheep receive fresh water ad libitum and food supplements throughout the year.The sheep in the entire farm are dewormed with 1% ivermectin (0.2 mg/kg BW subcutaneously) and albendazole (4 mg/kg BW orally) every 3 months.

Table 1 .
Cases positive for Nematodirus spp. in sheep diagnosed by Centrifugal Flotation and McMaster tests in the Veterinary Parasitology Laboratory of the Campus of Biological and Agricultural Sciences of the Universidad Autónoma de Yucatan.
that were introduced in flocks to the state of Yucatan.Only the sheep reported in the present case was born in the state of Yucatan and at the time of taking the fecal sample, it eliminated 100 EPG of Nematodirus spp.*Reported case, **order strongylida, EPG: eggs per gram of feces