Oestrosis: Parasitism by Oestrus ovis

Small Ruminant Research 181 (2019) 91–98

Contents lists available at ScienceDirect

Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres

Oestrosis: Parasitism by Oestrus ovis

T

⁎

M.J. Gracia , M. Ruíz de Arcaute, L.M. Ferrer, M. Ramo, C. Jiménez, L. Figueras Animal Pathology Department, Instituto Agroalimentario de Aragón-IA2 (Universidad de Zaragoza-CITA), Veterinary Faculty of Zaragoza, C/Miguel Servet 177, 50013 Zaragoza, Spain

A R T I C LE I N FO

A B S T R A C T

Keywords: Myiasis Oestrus ovis Sheep Goats

Oestrus ovis (Linnaeus 1761) is a parasite of sheep and goats, in which the fly larvae are obligatory parasites of nasal and sinus cavities. Oestrosis is endemic in hot and dry regions, especially in Mediterranean areas of Europe, Africa and America. Infected animals firstly suffer from fly strike, when adult flies inject first stage larvae on their nostrils and secondly, hosts will suffer from nasal-sinus myiasis with varying clinical respiratory signs. The disturbance caused to small ruminants while grazing and the effects during development of larvae can have severe consequences on livestock production. The evolution of O. ovis depends on the weather; the parasite is very well-adapted to their environment, being able to undergo hypobiosis either inside or outside the host, according to the climatic environmental conditions and seasonality. Understanding the epidemiology and life cycle of O. ovis is crucial to design effective control measures of this myiasis. Moreover, O. ovis infestation is considered a zoonosis; it causes ophthalmomyiasis in man in many parts of the world. The present article focuses on describing the main information about this parasite gathered in the last 20 years.

1. Introduction Myiasis is the infestation of living vertebrate animals and humans by the larvae of certain fly species (Diptera), which feed on the host’s living or dead tissues and body fluids. Oestrosis is a cavitary myiasis caused by the larvae of the fly Oestrus ovis (Linnaeus 1761, Diptera, Oestridae), which are obligate parasites (Zumpt, 1965). The sheep nose bot fly is a cosmopolitan parasite with economic importance particularly in hot and dry regions. It is especially widespread in Mediterranean countries of Europe and Africa, such as Italy (Caracappa et al., 2000; Scala et al., 2001, 2002), southern France (Yilma and Dorchies, 1991; Dorchies et al., 2000), Greece (Papadopoulos et al., 2001, 2006, 2010), Spain (Alcaide et al., 2003, 2005a; Gracia et al., 2010), northern Jordan (Abo-Shehada et al., 2000, 2003) and also in the warmer southern parts of the United Kingdom (Goddard et al., 1999), southwestern Germany (Bauer et al., 2002) and Mexico (Murguía et al., 2000). These studies show a high prevalence of oestrosis in small ruminants, between 30–91% and 18–75% in sheep and goats respectively. Adult females larviposit first-instar larvae directly into the nostrils of sheep and goats and their development in the nasal-sinus cavities can cause severe clinical signs including rhinitis, frequent sneezing, nasal discharge, breathing difficulties and emaciation, which together with the annoyance caused by the adult flies, may lead to significantly reduced animal production, causing losses of meat, wool and milk

⁎

production (Dorchies and Alzieu, 1997). Thus due to its high prevalence and the severity of the infection, it is necessary to treat infected small ruminants to prevent the effects of this parasitism. The larvae have also been found in other hosts and several cases of ophthalmic and nasopharyngeal myiasis have been reported in humans (Panadero-Fontán and Otranto, 2015) and dogs (Lucientes et al., 1997). In endemic areas, human cases of ocular affections are likely to occur more frequently than currently reported, because many benign, uncomplicated cases remain unreported (Panadero-Fontán and Otranto, 2015).The present article focuses on describing the main information about this parasite and data on biology, prevalence, pathology, immunology, diagnosis, treatment and control of O. ovis in the last 20 years is reviewed. 2. Morphological and biological traits Adult flies are about 12 mm long. The body is greyish-brown, with many small black spots on the thorax, which is covered with fine, light brown hair. They have rudimentary and non-functional oral mouthparts and are unable to feed (Fig. 1). Thus, the lifetime of adults must be short (from 2 to 4 weeks). Females emerge from the puparium with fully developed eggs ready for fertilization and their large eyes facilitate the localization of potential hosts, as well as suitable mates (AnguloValadez et al., 2010). In the temperate climate, adult flies are active from March-June to September-November depending on the area. In

Corresponding author. E-mail address: [email protected] (M.J. Gracia).

https://doi.org/10.1016/j.smallrumres.2019.04.017 Received 24 January 2019; Received in revised form 20 March 2019; Accepted 27 April 2019 Available online 28 April 2019 0921-4488/ © 2019 Elsevier B.V. All rights reserved.

Small Ruminant Research 181 (2019) 91–98

M.J. Gracia, et al.

Fig. 1. Adult fly of Oestrus ovis. Adult flies have rudimentary and non-functional oral mouthparts and are unable to feed.

countries with a warmer climate, adults are active during the wetter months (Yilma and Dorchies, 1991; Scala et al., 2002; Alcaide et al., 2003; Gracia et al., 2006). Mating of adult flies and larviposition occur on warm, sunny and windless days. Although temperature seems to be the main factor determining fly activity, wind and solar irradiance also play important roles. Fly strikes occur at temperatures greater than 20 °C, but mainly between 25 and 28 °C and from 116 to 838 W m−2 of solar irradiance. Few or no strikes are seen under moderate or strong wind, but can occur in a wide range of relative humidity. Likewise, movement of the animals is very important in stimulation of the flies (Cepeda-Palacios and Scholl, 2000a). O. ovis are larviparous and during their life span, female flies deposit up to 500 larvae into the nostrils or eyes of their hosts. The larva 1 (L1) is spindle-shaped, about 1 mm long and 0.36 mm wide. It bears two heavy buccal, bull's horn-shaped hooks (Fig. 2a), in the ventral face of the body shows rows of spines and in the last segment bears 20–24 ventrally bent, "cat's claws"-shaped hooks (Fig. 2b) (Giannetto et al., 1999). As soon as the laying has been carried out, L1 penetrate and go to nasal cavities, its structures avoid being expelled by host sneezing and help to move quickly into nasal passages (nasal septum, turbinates, and the ethmoid bone). It has been observed that larvae invade and spread through the entire nasal cavities (there is no larval aggregation) (Yacob et al., 2004b). The L1 grow to second instar (L2); it is 3–12 mm long and the size of their hooks and spines is reduced, not being therefore able to be expelled sneezing (Giannetto et al., 1999; AnguloValadez et al., 2010). The L2 migrates to the frontal sinuses and horn cavities and moult to third instar larvae (L3). The L3 is longer than 20 mm and it is provided with large hooks, stout spines and dorsal plates (Fig. 3) which serve to favour its gradual descend in the nasal cavities to the outside environment and perhaps its subsequent sinking into the soil for a few centimetres to change into a pupa (Giannetto et al., 1999). The larvae, in close contact with mucosa of the host, play an essential role in accumulating nutrients for the subsequent pupal and adult free-living stages. Larval feeding activity involves secretion of enzymes into the upper respiratory mucosal substrate, where they participate in extracorporeal pre-digestion. The biomolecules excreted/ secreted by the larvae and those in the larval salivary gland degrade the substrate into smaller units that are then swallowed to support larval growth and development. Specifically, larval enzymes (mainly serine proteases) are able to degrade mucosal and plasmatic components such as mucin, albumin and immunoglobulin G (Tabouret et al., 2003b; Angulo-Valadez et al., 2007b, 2010). There are no qualitative changes in the protease profile among the three instars, and the production is

Fig. 2. First instar larva. The first segment bears two oral chitinous hooks (a) and in the last segment, cat´s claws- shaped hooks (b).

Fig. 3. Ventral view of the third instar larva (courtesy of Dr Daniel MartínVega).

related to the increasing larval body size or nutrient requirements and their need to acquire reserves for the nonparasitic stages (AnguloValadez et al., 2010). The growth patterns of O. ovis larvae were estimated by Cepeda-Palacios et al. (1999). Larval weight increases from 0.23 mg in L1 larvae to 49 mg in late L2 larvae. The highest increase in weight occurs after the L2–L3 moult, especially during the early L3 period, when larvae acquire about 45% of the average mature weight (518 mg). Low larval weight can compromise the survival of pupal and adult stages; the estimated critical weight for mature larvae is 280 mg; below this value, larvae are expected to produce non-viable adult flies (Cepeda-Palacios et al., 2000). When the larvae have attained their

92

Small Ruminant Research 181 (2019) 91–98

M.J. Gracia, et al.

ruminants. Inside the host the L1 seem to have the capacity to adjust, pause or continue their developmental cycle during the parasitic phase, depending on the particular climatic conditions, such as temperature and humidity. The moults to L2 and L3 take place when the weather conditions are good enough for external pupation and adult fly activity. Hypobiotic period is usually characterised by the huge predominance of L1 instar. However, arrested and non-arrested L1 cannot be distinguished morphologically, so patterns of arrest must be inferred from overall larval stage structure, with higher L1 relative levels indicating slowed development and consequent accumulation. Dorchies and Alzieu (1997) suggested that L1 proportions above 80% indicate hypobiosis. Higher levels of L1 in relation to total larval burdens in winter, with a pronounced decrease in spring, are proposed in some regions to be due to hypobiosis. The highest percentages of L1 (above 80%) coincident with the absence or lowest percentages of second and third instars supported the idea that development into second and third instars was not occurring, and first instars were in diapause (Yilma and Dorchies, 1991; Abo-Shehada et al., 2000; Dorchies et al., 2000; Scala et al., 2002; Suarez et al., 2004; Paredes-Esquivel et al., 2012). In other regions such as Tunisie (Kilani et al., 1986) and Sicily (Caracappa et al., 2000) no hypobiosis period could be proved because the larval proportions are similar throughout the year. Their combined values show uniform percentage of L3, usually about 25%, suggesting a year-round development, additionally L1 are always higher than 35% and lower than 65%. A situation intermediate, where there is nor diapause nor fast development, were found in other surveys (Scala et al., 2001; Alcaide et al., 2003; Gracia et al., 2010; Papadopoulos et al., 2010). In these, the three instars were observed throughout the year (L1 higher than 55% and lower than 80%), although L3 was less frequent. The presence of L3 could indicate that development is not completely inhibited and the parasite continues to mature throughout the year. However, it could also be that L3 are not expelled, but remain arrested at this instar in the host (Gracia et al., 2010). An external hypobiotic period also has been observed. Biggs et al. (1998) indicated that the duration of pupation was variable; an extended pupation time is an efficient way to avoid the emergence of adults during adverse climatic conditions allowing adults flies to wait for the best time for emergence, mating and larviposition. This strategy may be attributable to warmer winter areas more than to localities in which overwintering is reported. This phenomenon has also been pointed out by Abo-Shehada et al. (2000) in northern Jordan. Definitely, the development of O. ovis is closely related to season and the number of annual cycles depends on particular climatic conditions (Tabouret et al., 2001a). Thus, in Spain (Alcaide et al., 2003), Jordan, (Abo-Shehada et al., 2003) and Greece (Papadopoulos et al., 2010) prevalence and larval abundance were both highest at warmer times of year, with twin peaks in spring and autumn. In France, however, three peaks were present, in spring, summer and autumn (Yilma and Dorchies, 1991). Finally, ovine surveys in other Mediterranean regions, such as Sardania (Scala et al., 2001), Tunisia (Kilani et al., 1986) and Sicily (Caracappa et al., 2000), reported the existence of a long favourable developmental period practically the entire year.

Fig. 4. Third instar larva, pupa and adult fly. It is observed the smaller size of the adult fly in relation to L3.

maximal growth, they migrate back to the nasal passages (L3 have spines and hooks useful for crawling outside), and they are expelled by the sneezing of the host onto the ground where they pupate (Fig. 4). The pupa has a cylindrical shape of 15 mm in length and 5 mm in width (Dorchies and Alzieu, 1997). Intrapuparial metamorphosis occurs over 19–27 days, after which flies emerge (Cepeda-Palacios and Scholl, 2000b). The lowest temperatures for the development of the O. ovis flies are 12.1 °C for males and 11.5 °C for females (Breev et al., 1980). According to Zumpt (1965), there is an asynchronous development, and few L3 larvae develop simultaneously. Slowing development allows larvae to survive overcrowding of too many larvae in a limited space. For this reason, the larval population profile involves many L1, fewer L2 and even fewer L3. When the percentage of L3 increases, mature L3 have to be expelled and cannot stay in the nasal cavities due to their size and to the intense local hypersensitive reaction. It has to be assumed that if there is a great number of L3 in the nasal cavities, there will be numerous L1 layed by adults two months later (Tabouret et al., 2001a; Angulo-Valadez et al., 2011). When the percentages of each of the three instars found in nasal cavities are similar, several generations are produced during that period and are indicative of rapid larval development and of many emergences of adults from the pupa (Tabouret et al., 2001a). The evolution of O. ovis is strongly influenced by climatic conditions. The length of the parasitic portion of the life cycle is quite variable, lasting from 3 to 4 weeks to several months depending on the season and climatic conditions (Zumpt, 1965). When temperature is above 12 °C larval activation begins, showing the highest activity at 25–28◦C (Angulo-Valadez et al., 2010). Under environmentally favourable conditions, larval development inside the host lasts 25–35 days. However, in temperate areas where the winter is too cold for pupariation, or during the hot and dry season of tropical countries, parasites reduce their metabolism, remaining quiescent in the host in a hypobiotic or diapause stage for up to 9 months (Dorchies et al., 2006). Arrested development is considered as a form of adaptation to local climate and hypobiosis may be observed either inside or outside small

3. Prevalence and risk factors O. ovis is found worldwide and oestrosis is endemic in hot and dry regions. It is especially widespread in Mediterranean areas of Europe, Africa and America. Table 1 shows the results of studies conducted in some of these areas. These studies show a high prevalence and larval burden of O. ovis in sheep and goats. Despite the fact that both sheep and goats can act as hosts of this parasite, prevalence and larval burdens are generally higher in sheep than in goats after either natural or artificial infestation (Abo-Shehada et al., 2000, 2003; Dorchies et al., 2000; Alcaide et al., 2003, 2005a; Papadopoulos et al., 2001, 2006). Goats might possibly have lower levels of infestation than sheep as a 93

Small Ruminant Research 181 (2019) 91–98

M.J. Gracia, et al.

Table 1 Prevalence and larval burdens of O. ovis in different areas. Countrie

Average anual temperature (º C)

Specie

Prevalence

Mean number of larvae per infested animal

Hypobiosis

Reference

France (south) France (southeast)

12.7

Sheep (n = 555 heads) Sheep (n = 631 heads) Goats (n = 672 heads) Sheep (n = 397 serum) Goats (n = 335 serum) Sheep (n = 292 heads) Goats (n = 158 heads) Sheep (n = 477 heads) Goats (n = 1590 serum) Goats (n = 80 heads) Sheep (n = 120) Sheep (n = 554 heads) Sheep (n = 841 heads) Sheep (n = 566 heads) Sheep (n = 443 heads) Sheep (n = 417 heads) Goats (n = 520 heads) Sheep (n = 689 serum) Sheep (n = 1497 serum) Sheep (n = 117 heads)

65%

24.8

Yes

Yilma and Dorchies, (1991)

43.4%

10.86

Yes

Dorchies et al. (2000)

28.4%

5.35

Yes

Dorchies et al. (2000)

48.6%

_

Papadopoulos et al. (2006)

17.9%

_

Papadopoulos et al. (2006)

Greece

Spain (southwest)

Spain (northeast) Spain (Balearics) Italy (Sicily) Italy (Sardinia)

Jordania (north)

14.5

15.0

16.0

14.7 16.1 18.4 16.2

18.3

Mexico

26.0

Germany (southwest) Argentina (South)

8.3 17.0

43.2%

10.33

Insufficient evidence

Papadopoulos et al. (2010)

75.9%

6.98

Insufficient evidence

Papadopoulos et al. (2010)

71.1%

18.5

Slower maduration

Alcaide et al. (2003)

_

Alcaide et al. (2005b)

46.04% 34.94%

3.9

Insufficient evidence

Alcaide et al. (2005b)

84.2%

37.9

Slower maduration

Gracia et al. (2010)

46.03%

10.83

Yes

Paredes-Esquivel et al. (2012)

55.8%

9.4

No

Caracappa et al. (2000)

91%

19

Slower maduration

Scala et al. (2001)

73.8%

6.07

Yes

Scala et al. (2002)

Yes

Abo-Shehada et al. (2000)

58% 24%

Abo-Shehada et al. (2003)

30.6%

_

Murguía et al. (2000)

50%

_

Bauer et al. (2002)

Yes

Suarez et al. (2004)

84.07

6.1

period there is little or no congestion of the nasal septum and turbinates (Angulo-Valadez et al., 2010). The pathogenesis of O. ovis infection is partially due to the mechanical trauma associated with the movement of larvae and irritation from cuticular spines and oral hooks, but mainly caused by biomolecules (enzymes and antigens) excreted or secreted by the larvae onto the mucosa that induce a hypersensitivity immune reaction (Dorchies et al., 1998, 2006; Jacquiet et al., 2005; Angulo-Valadez et al., 2011). The severity of pathogenesis is partially a consequence of the strong trophic activity of L2, but mainly of L3 larvae; when larval development increases not only does protein production in excreted/secreted products increase, but proteolytic activity is augmented (Tabouret et al., 2003b). Cuticle and excreted/secreted antigens have been identified and larval salivary antigens, in particular, have been found to strongly stimulate the immune system of sheep and goats (Tabouret et al., 2001b; Angulo-Valadez et al., 2009). The immune signalling and responses (cellular and humoral) derive from the recognition and processing of larval molecules at the mucosal level. Immune reactions involve the recruitment of cells (mast cells, eosinophils, macrophages, and T and B lymphocytes) and the secretion of immunoglobulins, suggesting a type Th2 immune response (AnguloValadez et al., 2011). The immune response may have an inhibitory effect on O. ovis larval growth, delaying development (Frugère et al., 2000; Angulo-Valadez et al., 2007a); nevertheless, previous exposure of animals to O. ovis larvae does not protect, neither against the establishment of re-infection with larvae nor against larval development

result of their avoidance behavioural responses; goats appear to be more sensitive to the irritation of ovipositing flies than sheep and may more effectively avoid larvae laying by adult flies (Abo-Shehada et al., 2003; Papadopoulos et al., 2006). Another factor may be that O. ovis larvae from sheep may be poorly adapted to goats (Dorchies et al., 1998). Additionally, there is some evidence that a caprine strain of O. ovis might exist with a much smaller distribution and lower prevalence compared to those of the ovine strain (Dorchies et al., 1998; Papadopoulos et al., 2006). Despite sheep and goats of all ages being affected by oestrosis, prevalence increases with age in both sheep (AboShehada et al., 2000; Murguía et al., 2000; Papadopoulos et al., 2010) and goats (Abo-Shehada et al., 2003). Flock size was found as risk factor; larger flocks were more likely to be seropositive than smaller ones (Bauer et al., 2002; Alcaide et al., 2005b). Likewise, nose colour was not found to be a significant predictor of infection (Papadopoulos et al., 2010), in contrast to previous suggestions that dark nosed animals were more likely to be infected (Murguía et al., 2000). 4. Pathogenesis and immune response Important effects are due to the activity of the adult flies. When they approach sheep and goats to deposit larvae the animals panic, stamp their feet, bunch together and press their nostrils into each other’s fleeces and against the ground. Rapidly after the L1 deposition, the migration of O. ovis larvae in the nasal cavities of the host induces an inflammatory response due to the parasite hooks and spines. During this 94

Small Ruminant Research 181 (2019) 91–98

M.J. Gracia, et al.

evade defensive attacks from the host. This may be a reason why poor acquired immunity develops over the lifespan of sheep and goats after several natural exposures to O. ovis. Immunosuppression may be orchestrated by L1 as a survival mechanism. An overstimulation of the immune response may lead to a ‘self-cure’ phenomenon after an important reinfection such as that which occurs in gastrointestinal parasite infections. This would mean the elimination of all L1 present in nasal cavities; therefore, modulating the immune response is important for L1 larvae (Angulo-Valadez et al., 2011). The concomitant infection with gastrointestinal nematodes and dipteran fly larvae such as O. ovis is a common phenomenon. Studies of the relationship between O. ovis and helminth co-infections have revealed that there are antagonist interactions between O. ovis larvae and Trichostrongylus colubriformis (Yacob et al., 2002, 2004a, 2006; Silva et al., 2012) and Haemonchus contortus (Dorchies et al., 1997a; Terefe et al., 2005; Yacob et al., 2008; Silva et al., 2012). The presence of O. ovis larvae is correlated with significant reductions in nematode egg excretion, female worm fecundity and worm burdens. In addition, animals infected with O. ovis seem to be more tolerant to the pathogenic effects of haemonchosis (Terefe et al., 2005; Yacob et al., 2008; Silva et al., 2012). Thus, O. ovis infestation stimulates the immune response, towards the upper respiratory tract and digestive mucosae (Dorchies et al., 1997a; Yacob et al., 2002, 2004a, 2006; Terefe et al., 2005). Yacob et al. (2004b) reported the presence of an early and high eosinophilia and migration of the same cellular components into the abomasal and intestinal mucosae in the absence of nematodes in the gut. It may be assumed that the mechanism involved in the interaction was non-specific and probably related to enhanced recruitment of circulating eosinophils and/or their products towards the gut mucosa in the presence of O. ovis that created unfavourable environment to the nematodes (Yacob et al., 2002; 2006, 2008; Terefe et al., 2005; Silva et al., 2012). The negative interactions between O. ovis larvae infection and nematodes of the digestive tract are of transient nature and disappear when O. ovis are expelled (Jacquiet et al., 2005; Yacob et al., 2006). However, the infection of the digestive tract with nematodes did not modify the biology of Oestrus populations (Dorchies et al., 1997a; Yacob et al., 2002, 2004a, 2008; Terefe et al., 2005). Immunization trials against O. ovis have been performed in sheep (Frugère et al., 2000; Angulo-Valadez et al., 2007a) and the results suggested that at least a partial regulation of O. ovis larvae populations could occur within the sheep. In both experiments, no significant effect of immunization on larval establishment was observed but provided an inhibitory effect on larval growth. Reduced weight of O. ovis mature third instars (< 280 mg) may compromise subsequent survival of the free stages (Cepeda-Palacios et al., 2000).

after re-infections following antiparasitic treatments (Jacquiet et al., 2005). O. ovis is considered a parasite with proinflammatory activity; this is especially true in the case of L2 and L3 larvae. O. ovis induces massive recruitment and degranulation of mast cells. This was related to larval development and infection sites; numbers of mast cells were found to sharply increase from the septum to the ethmoid sinus (Yacob et al., 2002, 2004a, 2004b; Tabouret et al., 2003a). In naturally infected sheep the mean number of mast cells and eosinophils were twice those in parasite-free animals (Dorchies et al., 1998). Mast cells may limit the larval population inside the host (Jacquiet et al., 2005); releasing hydrolytic enzymes which may act on the cuticle of the parasite (Dorchies et al., 2006). The presence of O. ovis larvae is also related to a strong activation of eosinophils (Yacob et al., 2002, 2004a, 2004b; 2006; Tabouret et al., 2003a; Jacquiet et al., 2005), however, no relationship between the number of eosinophils in mucosae and larval burden or development was apparent (Angulo-Valadez et al., 2011). Nevertheless, the reaction was not only local but regional, a significant accumulation of eosinophils in the tissues of the trachea, bronchi and lungs was observed even though O. ovis was not present there (Yacob et al., 2004b). There was an increased number of circulating blood eosinophils in the animals infected with O. ovis larvae and eosinophilia was significantly correlated with the establishment rate of O. ovis in infected animals (Yacob et al., 2002, 2004b, 2008; Terefe et al., 2005). Likewise, macrophages and mononuclear cells (B and T lymphocytes) were more numerous in the mucosa in infected than in control animals (Tabouret et al., 2003a; Angulo-Valadez et al., 2011). Infected lambs secreted specific nasal mucous IgG and this local humoral response has been found to be clearly associated with O. ovis infection (Tabouret et al., 2003a; Jacquiet et al., 2005) and high local IgG responses were negatively correlated with larval establishment and/or development inside the host. Such correlations were observed in some sheep families but not in others, which suggests that some genetic control of resistance and susceptibility mediated by IgG antibodies may be involved (AnguloValadez et al., 2008). Values of total serum IgG level in infected animals were significantly higher than those of non-infected (Terefe et al., 2005) and systemic IgG response was highly correlated with prevalence of infection (Angulo-Valadez et al., 2009) and number of O. ovis larvae (Alcaide et al., 2005b; Silva et al., 2012). IgG seroconversion is usually reached 2–4 weeks post first infection (Frugère et al., 2000; Tabouret et al., 2001b; Yacob et al., 2002; Angulo-Valadez et al., 2007a; Silva et al., 2012) or even later (6–10 weeks), when only L1 or very low numbers of larvae are present (Terefe et al., 2005; Angulo-Valadez et al., 2009). The stage of larval development has a significant impact on the humoral immune response over the course of a season, suggesting different antigenic characteristics for each larval stage (Alcaide et al., 2005b). The development of first instars developed to second and third instars stimulates the production of IgG antibodies; the highest levels are observed during the development of L2 and L3 larvae (Dorchies et al., 1998; Scala et al., 2002; Tabouret et al., 2003a; Suarez et al., 2004, 2005; Angulo-Valadez et al., 2011; Silva et al., 2012). Suarez et al. (2005) observed that the number of O. ovis L2 larvae and serum IgG levels were positively correlated, and that a seasonal reduction of IgG antibodies was associated with the end of the larval growth period or beginning of the diapausal season. During the diapause, first instars reduce both their activity and metabolism and remain quiescent in the nasal cavities, so they may therefore present either no antigen or only very low levels of antigen to the immune system (Goddard et al., 1999). Likewise, specific IgG was found to have almost disappeared 160 days after the administration of an effective antiparasite treatment (Dorchies et al., 2003). Systemic production of IgM was positively correlated with the number of O. ovis first instars hosted (Suarez et al., 2005). The highest values were recorded justly during the diapause indicating that this increment was not associated to the active metabolism of O. ovis larvae. Host–O. ovis relationships include strategies of immunostimulation (Tabouret et al., 2001b, 2003b) and immunosuppression (Jacquiet et al., 2005; Tabouret et al., 2003b) to

5. Clinical manifestations Infected animals demonstrate two clinical phases, fly strike and myiasis, while there are no clinical signs during L1 hypobiosis period. Fly activity affects sheep and goat behaviour making the animals get nervous and gathering close together, keeping their noses deep inside the fleece of the neighbours or close to the ground during displacements, in an attempt to avoid the larvipositing female flies. During this period only a small amount of mucus is observed. A few weeks after larviposition, (L2 and L3 development) nasal discharge and sneezing become more evident and frequent. Animals are agitated and the nasal discharge, which is initially serous, becomes sero-mucous, mucopurulent and eventually, in the most severe cases, purulent (Fig. 5). There is presence of eosinophils in the nasal discharge and also in mucosae. Blood strands can be found in the nasal discharges, due to the mechanical action of the larvae. In young animals that are infested for the first time, the reaction is more intense and the nasal secretions are more profuse and serous (Fig. 5). The amount of nasal discharge is not related to the number of larvae, but appears to be related to the individual susceptibility and also to interactions with bacteria. In 95

Small Ruminant Research 181 (2019) 91–98

M.J. Gracia, et al.

in the morning, the movement produced when they wake up makes the nasal discharge (uni or bilateral) and sneezing more evident and frequent. Moreover, blood strands can be seen in the nasal discharges. It is also observed that more parasitized animals walk slower and show clear signs of dyspnoea that leads them to breathe through their mouth. In addition, the farmer/veterinarian may find L3 larvae which have been expelled by the animals inside the troughs and water troughs. Finally, the improvement of the animals after the application of an antiparasitic treatment occasionally allows us to confirm the process. Different tests are used to study the serological prevalence and diagnosis of O. ovis, in special the enzyme-linked immunosorbent assay (ELISA) (Papadopoulos et al., 2001; Tabouret et al., 2001b; Suarez et al., 2004; Jacquiet et al., 2005; Angulo-Valadez et al., 2007a, 2009; Silva et al., 2012). The stage of larval development has a significant impact on the humoral immune response over the course of a season and the highest values were obtained when their homologous larval stage was present in the host (Alcaide et al., 2005b). Suarez et al. (2005) observed that the number of O. ovis L1 larvae and serum IgM levels were positively correlated, as were L2 larvae and IgG levels, and that a seasonal reduction of IgG antibodies was associated with the end of the larval growth period or beginning of the diapause season. Positive values of IgG antibodies only indicate a previous contact to the parasitic antigens, and not necessarily the presence of current infection. High levels of specific IgG antibodies could persist after the expulsion of any remaining mature larvae or after larvicidal treatment (Alcaide et al., 2005b). Moreover, detection of infected animals is difficult in winter samples, resulting in false negatives; hypobiosis of L1 larvae leads to the loss of metabolic and migratory activities and hence a decrease of antigen stimulation. In southwest of Spain, the ELISA test using L1 antigen during winter and L2 antigen in summer may be used for ovine oestrosis immunodiagnosis (Alcaide et al., 2005b). Although serological methods are not useful for diagnosis they have proved to be interesting for regional surveys. Recently, the usefulness of semi-nested PCR and rhinoscopy in the diagnosis of oestrosis was evaluated by Ípek and Altan (2017).

Fig. 5. Clinical signs in adult (a) and young sheep (b). Sero-mucous discharge is common in adult sheep while the reaction in young animals is more intense and the nasal secretions are more profuse and serous.

addition to these local effects, lung abscesses and interstitial pneumonia develops during the course of ovine oestrosis. It may be assumed that lung abscesses are related to pyogenic focus in the nasosinusal area and the pathology of interstitial pneumonia is probably caused by aspirated bacteria, eosinophils or larval antigens. Considerable numbers of eosinophils and mast cells have been observed in the lung parenchyma, mainly in the peribronchial region (Dorchies et al., 1998). Eventually, the nasal passages become obstructed by mucus and dust leading to a difficulty of breathing, which reduces grazing activity and rumination time and commonly results in negative nutritional effects such as general malnutrition, low performance and even death. Heavily infested animals may exhibit neurological symptoms: including ataxia, vertigo, nystagmus and amaurosis as well as epistaxis (Dorchies and Alzieu, 1997). The sinusal mucosa of infected animals is thickened and the infection induces hyperplasia and metaplasia in the epithelium in the nasal, ethmoid and sinus cavities (Tabouret et al., 2003a). In some breeds of sheep, neoplastic tumors (adenocarcinoma of the pituitary mucosae, 4–5 cm wide) might be found (Angulo-Valadez et al., 2010).

7. Treatment Currently, control measures against this parasite depend on treatment with closantel and macrocyclic lactones (ivermectin and moxidectin). The efficacy of other macrocyclic lactones members (doramectin and eprinomectin) has also been tested in naturally infected sheep. Table 2 shows the results of studies conducted to assess the efficacy of different products against O. ovis larvae. In general, all the products present a high efficacy against O. ovis. The lower efficacy against L1 than against L2 or L3, are consistent with the biology of L1; the L1 have limited feeding and are therefore less susceptible to systemic parasiticides (Rugg et al., 2012). However, when L1 start to develop and increase their feeding, ingest more dose of treatment and die (Martínez-Valladares et al., 2013). Finally, oral administration of neem seeds and leaves (Azadirachta indica A. Juss) had not effect on larval survival, but tended to delay larval development in experimentally O. ovis infected sheep (Cepeda-Palacios et al., 2014). In any case, the use of products against O. ovis must be in accordance with European legislation (Regulation, 2019/6/EU repealing Directive, 2001/82/ EURegulation /6/EU repealing Directive /82/EU, 2019Regulation, 2019/6/EU repealing Directive, 2001/82/EU). Due to close relationship between climatic conditions and the development of O. ovis, a thorough knowledge of parasite epidemiology and life cycle is crucial to design efficient control strategies. O. ovis has a relatively short free-living life cycle outside of the host. Therefore, it is necessary to know when the parasitic period occurs in order to prevent the clinical signs and economic losses caused by this parasite. The length of this parasitic portion of the life cycle is quite variable: it ranges from a few weeks to several months depending on the season and climatic conditions. For this reason the right time of treatment must

6. Diagnosis The diagnosis of oestrosis is commonly and easily done by symptoms (fly strike and myiasis) and during the necropsy of the animals through the detection of larval stages. Fly activity affects animal behaviour. During the fly strike, the animals panic, stamp their feet, bunch together and press their nostrils into each other’s fleeces and against the ground. When the flies are not active, during the early morning and late afternoon, animals spread widely over the field. Oestrosis is a collective pathology, usually affecting a high number of individuals in the flock and this aspect differentiates it from the main upper respiratory tracts pathologies, which are more commonly individual. The symptoms are more evident early in the morning; the animals accumulate secretions during the night and 96

Small Ruminant Research 181 (2019) 91–98

M.J. Gracia, et al.

Table 2 Results of studies conducted to assess the efficacy of different products against O. ovis larvae. Active ingredient

Closantel Ivermectin

Moxidectin

O. ovis

Dose mg/Kg b.w. 10 0.2

0.2

L1

Oral Subcutaneous oral Subcutaneous

oral

Doramectin Eprinomectin

1

Subcutaneous

1

Subcutaneous

0.2 1

Intramuscular Pour-on

L2

100% (Day 12)

100% (Day 80)

100%

Intestinal strongyles

Liver flukes

Reference

6-8 weeks No evidence < 6 weeks

– + + +

+

Dorchies et al. (1997b) Dorchies et al. (1997b) Dorchies et al. (1997b) Lucientes et al. (1998)

+

–

Dorchies et al. (1996)

+

–

L3

100% 98% 100% 100% (Day 12) 80% 91% (Day 14) 65% (Day 35) 68% (Day 14) 0% (Day 35) 38% (Day 20) 90% (Day 28) 100% 100%

Persistent activity

100% (Day 12)

100% (Day 80)

80 days

+

Rugg et al. (2012)

+

Martínez-Valladares et al. (2013) Dorchies et al. (2001) Habela et al. (2006)

+ +

100%

– –

of larvae into the eyes, with patients often describing insects or small foreign bodies striking the eye before the occurrence of symptoms, which usually include acute pain, conjunctivitis, soreness, lachrymation and a sensation of foreign bodies moving in the eye. Mucopurulent conjunctivitis can also occur as a consequence of secondary infections (Panadero-Fontán and Otranto, 2015). Sneezing and epiphora may also occur when larvae are sprayed into the nose and mouth (HemmersbachMiller et al., 2007).

be adapted to local climatic conditions. In areas where no period of hypobiosis was recorded (or only slowed development) in order to lower prevalence of this disease, it would be necessary to use a parasiticide effective against O. ovis for all routine parasite control treatments (Caracappa et al., 2000; Scala et al., 2001). In areas where O. ovis undergoes hypobiosis, strategic control of the parasite should be concentrated on the total elimination of overwintering larvae and large scale systematic treatments in late autumn or winter are considered efficient to control the disease (Scala et al., 2002; Gracia et al., 2006). The use of two strategic treatments per year; the first one in FebruaryMarch and the second in November (Alcaide et al., 2003) or at the beginning of the hypobiotic period (Paredes-Esquivel et al., 2012) also seems to be very efficient in O. ovis control. Paredes-Esquivel et al. (2012) recommended the use of persistent drugs during the flight activity to protect the animals from reinfection while less persistent drugs could be used at the beginning of the hypobiotic period. A preventive treatment should be applied with the aim to be effective against L1 since they do not produce the pathogenic actions described for L2 and L3. However, the efficacy of several systemic parasiticides seems to be lower against L1 than against L2 or L3. For this reason and according to veterinarian recommendations of our region, sheep breeders treat their animals at the beginning of the season when the larvae start to develop to L2 and L3 and when the symptomatology still it is not obvious. Thus, control measures should be closely adjusted to the climate in each area. However, the conflicting results reported on the number of generations in different countries and the interannual variations in oestrosis prevalence indicate the need of monitoring the disease to establish the appropriate timing of treatments. Paredes-Esquivel et al. (2012) hypothesize that lambs are better indicators of the seasonality of oestrosis than their older counterparts and propose that observing O. ovis infestations in young lambs can be used as an efficient early warning system of fly activity, to be applied in future control programs.

Conflict of interest statement The authors have nothing to disclose. References Abo-Shehada, M.N., Arab, B., Mekbel, R., Willians, D., Torgerson, P.R., 2000. Age and seasonal variations in the prevalence of Oestrus ovis larvae among sheep in northern Jordan. Prev. Vet. Med. 47, 205–212. Abo-Shehada, M.N., Batainah, T., Abuharfeil, N., Torgerson, P.R., 2003. Oestrus ovis larval myiasis among goats in northern Jordan. Prev. Vet. Med. 59, 13–19. Alcaide, M., Reina, D., Sánchez, J., Frontera, E., Navarrete, I., 2003. Seasonal variations in the larval burden distribution of Oestrus ovis in sheep in the southwest of Spain. Vet. Parasitol. 118, 235–241. Alcaide, M., Reina, D., Frontera, E., Navarrete, I., 2005a. Epidemiology of Oestrus ovis (Linneo, 1761) infestation in goats in Spain. Vet. Parasitol. 130, 277–284. Alcaide, M., Reina, D., Frontera, E., Navarrete, I., 2005b. Analysis of larval antigens of Oestrus ovis for the diagnosis of oestrosis by enzyme-linked immunosorbent assay. Med. Vet. Entomol. 19, 151–157. Angulo-Valadez, C.E., Cepeda-Palacios, R., Jacquiet, P., Dorchies, P., Prévot, F., AscencioValle, F., Ramírez-Orduña, J.M., Torres, F., 2007a. Effects of immunization of Pelibuey lambs with Oestrus ovis digestive tract protein extracts on larval establishment and development. Vet. Parasitol. 143, 140–146. Angulo-Valadez, C.E., Cepeda-Palacios, R., Ascencio, F., Jacquiet, P., Dorchies, P., Romero, M.J., Khelifa, R.M., 2007b. Proteolytic activity in salivary gland products of sheep bot fly (Oestrus ovis) larvae. Vet. Parasitol. 149, 117–125. Angulo-Valadez, C.E., Scala, A., Grisez, C., Prévot, F., Bergeaud, J.P., Carta, A., CepedaPalacios, R., Ascencio, F., Terefe, G., Dorchies, P., Jacquiet, P., 2008. Specific IgG antibody responses in Oestrus ovis L. (Diptera: Oestridae) infected sheep: Associations with intensity of infection and larval development. Vet. Parasitol. 155, 257–263. Angulo-Valadez, C.E., Cepeda-Palacios, R., Ascencio, F., Jacquiet, Ph., Dorchies, Ph., Ramírez-Orduña, J.M., López, M.A., 2009. IgG antibody response against salivary gland antigens from Oestrus ovis L. larvae (Diptera: Oestridae) in experimentally and naturally infected goats. Vet. Parasitol. 161, 356–359. Angulo-Valadez, C.E., Scholl, Ph., Cepeda-Palacios, R., Jacquiet, Ph., Dorchies, Ph., 2010. Nasal bots… a fascinating world!. Vet. Parasitol. 174, 19–25. Angulo-Valadez, C.E., Ascencio, F., Jacquiet, P., Dorchies, P., Cepeda-Palacios, R., 2011. Sheep and goat immune responses to nose bot infestation: a review. Med. Vet. Entomol. 25, 117–125. Bauer, C., Steng, G., Prévot, F., Dorchies, P., 2002. Seroprevalence of Oestrus ovis

8. Human infection Adult flies may eject larvae into human eyes, inducing an external opthalmomyiasis or more rarely, nasal or oral myiasis (Angulo-Valadez, et al., 2010). Mostly affected are shepherds and farmers from rural areas. In endemic areas, cases of ocular affections caused by O. ovis are likely to occur more frequently than currently reported since most cases are misdiagnosed or unreported, mainly because of the mild symptoms that they cause. The infestation occurs when female flies eject a stream 97

Small Ruminant Research 181 (2019) 91–98

M.J. Gracia, et al.

instar larvae of Oestrus ovis in naturally infected sheep. Vet. Parasitol. 193, 320–324. Murguía, M., Rodríguez, J.C., Torres, F.J., Segura, J.C., 2000. Detection of Oestrus ovis and associated risk factors in sheep from the central region of Yucatan, Mexico. Vet. Parasitol. 88, 73–78. Panadero-Fontán, R., Otranto, D., 2015. Arthropods affecting the human eye. Vet. Parasitol. 208, 84–93. Papadopoulos, E., Prévot, F., Jacquiet, P., Duranton, C., Bergeaud, J.P., Kalaitzakis, E., Dorchies, P., 2001. Seasonal variation of Oestrus ovis specific antibodies in sheep and goats mixed flocks in Greece. Vet. Parasitol. 95, 73–77. Papadopoulos, E., Prévot, F., Diakou, A., Dorchies, P., 2006. Comparison of infection of Oestrus ovis between sheep and goats kept in mixed flocks. Vet. Parasitol. 138, 382–385. Papadopoulos, E., Chaligiannis, I., Morgan, E.R., 2010. Epidemiology of Oestrus ovis L. (Diptera: Oestridae) larvae in sheep and goats in Greece. Small Rumin. Res. 89, 51–56. Paredes-Esquivel, C., del Río, R., Monerris, M., Borrás, D., Laglera, L.M., Miranda, M.A., 2012. The influence of sheep age group on the seasonal prevalence of oestrosis in the island of Majorca. Vet. Parasitol. 186, 538–541. Regulation (EU) 2019/6 of the European Parliament and of the Council of 11 December 2018 on veterinary medicinal products and repealing Directive 2001/82/EC. Rugg, R., Ferrer, L.M., Sarasola, P., Figueras, L., Lacasta, D., Liu, B., Bartram, D., 2012. Persistent efficacy of a long acting injectable formulation of moxidectin against natural infestations of the sheep nasal bot (Oestrus ovis) in Spain. Vet. Parasitol. 188, 330–336. Scala, A., Solinas, G., Citterio, C.V., Kramer, L.H., Genchi, C., 2001. Sheep oestrosis (Oestrus ovis Linne 1761, Diptera: Oestridae) in Sardinia, Italy. Vet. Parasitol. 102, 133–141. Scala, A., Paz-silva, A., Suarez, J.L., López, C., Díaz, P., Díez-Baños, P., Sánchez-Andrade Fernández, R., 2002. Chronobiology of Oestrus ovis (Diptera: Oestridae) in Sardinia, Italy: guidelines to chemoprophylaxis. J. Med. Entomol. 39 (4), 652–657. Silva, B.F., Bassetto, C.C., Amarante, A.F.T., 2012. Immune responses in sheep naturally infected with Oestrus ovis (Diptera: Oestridae) and gastrointestinal nematodes. Vet. Parasitol. 190, 120–126. Suarez, V.H., Busetti, M.R., Miranda, A.O., Prévot, F., Jacquiet, P., 2004. Epidemiology of Oestrus ovis infection of sheep in Argentina’s western Pampas. Parasite. 11, 405–410. Suarez, J.L., Scala, A., Romero, J.A., Paz-Silva, A., Pedreira, J., Arias, M., Diaz, P., Morrondo, P., Diez-Baños, P., Sánchez-Andrade, R., 2005. Analysis of the humoral immune response to Oestrus ovis in ovine. Vet. Parasitol. 134, 153–158. Tabouret, G., Jacquiet, P., Scholl, P., Dorchies, P., 2001a. Oestrus ovis in sheep: relative third-instar populations, risks of infection and parasite control. Vet. Res. 32, 525–531. Tabouret, G., Prévot, F., Bergeaud, J.P., Dorchies, Ph., Jacquiet, P., 2001b. Oestrus ovis (Diptera: Oestridae): sheep humoral immune response to purified excreted/secreted salivary gland 28 kDa antigen complex from second and third instar larvae. Vet. Parasitol. 101, 53–66. Tabouret, G., Lacroux, C., Andreoletti, O., Bergeaud, J.P., Hailu-Tolosa, Y., Hoste, H., Prévot, F., Grisez, C., Dorchies, Ph., Jacquiet, Ph., 2003a. Cellular and humoral local immune responses in sheep experimentally infected with Oestrus ovis (Diptera: Oestridae). Vet. Res. 34, 231–241. Tabouret, G., Bret-Bennis, L., Dorchies, P., Jacquiet, P., 2003b. Serine protease activity in excretory–secretory products of Oestrus ovis (Diptera: Oestridae) larvae. Vet. Parasitol. 114, 305–314. Terefe, G., Yacob, H.T., Grisez, C., Prévot, F., Dumas, E., Bergeaud, J.P., Dorchies, Ph., Hoste, H., Jacquiet, P., 2005. Haemonchus contortus egg excretion and female length reduction in sheep previously infected with Oestrus ovis (Diptera: Oestridae) larvae. Vet. Parasitol. 128, 271–283. Yacob, H.T., Duraton-Grisez, C., Prévot, F., Bergeaud, J.P., Bleuart, C., Jacquiet, Ph., Dorchies, Ph., Hoste, H., 2002. Experimental concurrent infection of sheep with Oestrus ovis and Trichostrongylus colubriformis: and related changes in the cellular responses of nasal and digestive mucosae. Vet. Parasitol. 104, 307–317. Yacob, H.T., Dorchies, Ph., Jacquiet, Ph., Bleuart, C., Prévot, F., Grisez, C., Bergeaud, J.P., Hoste, H., 2004a. Concurrent parasitic infections of sheep: depression of Trichostrongylus colubriformis populations by a subsequent infection with Oestrus ovis. Vet. Parasitol. 121, 297–306. Yacob, H.T., Jacquiet, Ph., Prévot, F., Bergeaud, J.P., Bleuart, C., Dorchies, Ph., Hoste, H., 2004b. Examination of the migration of first instar larvae of the parasite Oestrus ovis (Linne 1761) [Diptera: Oestridae] in the upper respiratory tract of artificially infected lambs and daily measurements of the kinetics of blood eosinophilia and mucosal inflammatory response associated with repeated infection. Vet. Parasitol. 126, 339–347. Yacob, H.T., Terefe, G., Jacquiet, P.H., Hoste, H., Grisez, C., Prévot, F., Bergeaud, J.P., Dorchies, P.H., 2006. Experimental concurrent infection of sheep with Oestrus ovis and Trichostrongylus colubriformis: effects of antiparasitic treatments on interactions between parasite populations and blood eosinophilic response. Vet. Parasitol. 137, 184–188. Yacob, H.T., Basazinew, B.K., Basu, A.K., 2008. Experimental concurrent infection of Afar breed goats with Oestrus ovis (L1) and Haemonchus contortus (L3): interaction between parasite populations, changes in parasitological and basic haematological parameters. Exp. Parasitol. 120, 180–184. Yilma, J.M., Dorchies, Ph., 1991. Epidemiology of Oestrus ovis in southwest France. Vet. Parasitol. 40, 315–323. Zumpt, F., 1965. Myiasis in Man and Animals in the Old-world. Ed. Butterworth, London, 225 pp.. .

infection in sheep in southwestern Germany. Vet. Parasitol. 110, 137–143. Biggs, H.C., Mcclain, E., Muller, G.L., Anthonissen, M., Hare, K.M., 1998. A prediction model for strike in the sheep nasal fly, Oestrus ovis, in Namibia. Prev. Vet. Med. 33, 267–282. Breev, K.A., Zagretdinov, R.G., Minar, J., 1980. Influence of constant and variable temperature on pupal development of the sheep bot fly (Oestrus ovis L.). Folia Parasitol. 27, 359–365. Caracappa, S., Rilli, S., Zanghi, P., Di Marco, V., Dorchies, P., 2000. Epidemiology of ovine oestrosis (Oestrus ovis Linné 1761, Diptera: Oestridae) in Sicily. Vet. Parasitol. 92, 233–237. Cepeda-Palacios, R., Scholl, P.J., 2000a. Factors affecting the larvipositional activity of Oestrus ovis gravid females (Diptera: Oestridae). Vet. Parasitol. 91, 93–105. Cepeda-Palacios, R., Scholl, P.J., 2000b. Intra-puparial development in Oestrus ovis (Diptera: Oestridae). J. Med. Entomol. 37 (2), 239–245. Cepeda-Palacios, R., Ávila, A., Ramírez-Orduña, R., Dorchies, Ph., 1999. Estimation of the growth patterns of Oestrus ovis L. larvae hosted by goats in Baja California Sur. Mexico. Vet. Parasitol. 86, 119–126. Cepeda-Palacios, R., Frugère, S., Dorchies, P., 2000. Expected effects of reducing Oestrus ovis L. mature larval weight on adult populations. Vet. Parasitol. 90, 239–246. Cepeda-Palacios, R., Servín, R., Ramírez-Orduña, J.M., Ascencio, F., Dorchies, P., AnguloValadez, C.E., 2014. In vitro and in vivo effects of neem tree (Azadirachta indica A. Juss) products on larvae of the sheep nose bot fly (Oestrus ovis L. Díptera: Oestridae). Vet. Parasitol. 200, 225–228. Dorchies, Ph., Alzieu, J.P., 1997. L´oestrose ovine: revue. Revue Méd. Vét. 148, 565–574. Dorchies, P., Cardinaud, B., Foumier, R., 1996. Efficacy of moxidectin as a 1% injectable solution and a 0.1% oral drench against nasal bots, pulmonary and gastrointestinal nematodes in sheep. Vet. Parasitol. 65, 163–168. Dorchies, Ph., Bergeaud, J.P., Nguyen, V.K., Morand, S., 1997a. Reduced egg count in mixed infection with Oestrus ovis and Haemonchus contortus: influence of eosinophils. Parasitol. Res. 83, 727–730. Dorchies, Ph., Alzieu, J.P., Cadiergues, M.C., 1997b. Comparative curative and preventive efficacies of ivermectin and closantel on Oestrus ovis (Linné 1758) in naturally infected sheep. Vet. Parasitol. 72, 179–184. Dorchies, P., Duranton, C., Jacquiet, P., 1998. Pathophysiology of Oestrus ovis infection in sheep and goats: a review. Vet. Rec. 142, 487–489. Dorchies, Ph., Bergeaud, J.P., Tabouret, G., Duranton, C., Prévot, F., Jacquiet, Ph., 2000. Prevalence and larval burden of Oestrus ovis (Linné 1761) in sheep and goats in northern Mediterranean region of France. Vet. Parasitol. 88, 269–273. Dorchies, Ph., Jacquiet, Ph., Bergeaud, J.P., Duranton, C., Prévot, F., Alzieu, J.P., Gossellin, J., 2001. Efficacy of doramectin injectable against Oestrus ovis and gastrointestinal nematodes in sheep in the southwestern region of France. Vet. Parasitol. 96, 147–154. Dorchies, Ph., Wahetra, S., Lepetitcolin, E., Prévot, F., Grisez, C., Bergeaud, J.P., Hoste, H., Jacquiet, Ph., 2003. The relationship between nasal myiasis and the prevalence of enzootic nasal tumours and the effects of treatment of Oestrus ovis and milk production in dairy ewes of Roquefort cheese area. Vet. Parasitol. 113, 169–174. Dorchies, P., Tabouret, G., Hoste, H., Jacquiet, P., 2006. Oestrinae host-parasite interactions. In: Colwell, D.D., Hall, M.J.R., Scholl, P.J. (Eds.), The Oestrid Flies. CABI Publishing p. 359. Frugère, S., Cota-Leon, L.A., Prévot, F., Cepeda-Palacios, R., Tabouret, G., Bergeaud, J.P., Duranton, Ch., Dorchies, Ph., Jacquiet, Ph., 2000. Immunization of lambs with excretory-secretory products of Oestrus ovis third-instar larvae and subsequent experimental challenge. Vet. Res. 31, 527–535. Goddard, P., Bates, P., Webster, K.A., 1999. Evaluation of a direct ELISA for the serodiagnosis of Oestrus ovis infections in sheep. Vet. Rec. 144, 497–501. Gracia, M.J., Lucientes, J., Peribáñez, M.A., Calvete, C., Ferrer, L.M., Castillo, J.A., 2006. Kinetics of Oestrus ovis infection and activity of adult flies. Parasite. 13, 311–313. Gracia, M.J., Lucientes, J., Peribáñez, M.A., Castillo, J.A., Calvete, C., Ferrer, L.M., 2010. Epidemiology of Oestrus ovis infection of sheep in northeast Spain (mid-Ebro Valley). Trop. Anim. Health Prod. 42, 811–813. Habela, M., Moreno, A., Gragera-Slikker, A., Gómez, J.M., Montes, G., Rodríguez, P., Alvinerie, M., 2006. Efficacy of eprinomectin pour-on in naturally Oestrus ovis infested merino sheep in Extremadura, South-West Spain. Parasitol. Res. 99, 275–280. Hemmersbach-Miller, M., Sánchez-Andrade, R., Domínguez-Coello, A., Hawari, A., PazSilva, A., Carranza, C., Pérez-Arellano, J.L., 2007. Human Oestrus sp infection, Canary Islands. Emerg. Infect. Dis. 13, 950–952. Ípek, D.N.S., Altan, S., 2017. Use of semi-nested PCR and rhinoscopy for the diagnosis of oestrosis. Small Rumin. Res. 150, 76–79. Jacquiet, P., Thi, T.T.N., Nouvel, X., Prévot, F., Grisez, C., Yacob, H.T., Bergeaud, J.P., Hoste, H., Dorchies, P.H., Tabouret, G., 2005. Regulation of Oestrus ovis (Diptera: Oestridae) populations in previously exposed and naive sheep. Vet. Immunol. Immunopathol. 105, 95–103. Kilani, M., Kacem, H., Dorchies, Ph., Franc, M., 1986. Observations sur le cycle annuel d´Oestrus ovis en Tunisie. Revue Méd. Vét. 137, 451–457. Lucientes, J., Ferrer-Dufol, M., Andres, M.J., Peribáñez, M.A., Gracia-Salinas, M.J., Castillo, J.A., 1997. Canine Myiasis by Sheep Bot Fly (Diptera: Oestridae). J. Med. Entomol. 34 (2), 242–243. Lucientes, J., Castillo, J.A., Ferrer, L.M., Peribáñez, M.A., Ferrer-Dufol, M., GraciaSalinas, M.J., 1998. Efficacy of orally administered ivermectin against larval stages of Oestrus ovis in sheep. Vet. Parasitol. 75, 255–259. Martínez-Valladares, M., Valcárcel, F., Álvarez-Sánchez, M.A., Cordero-Pérez, C., Fernández-Pato, N., Frontera, E., Meana, A., Rojo-Vázquez, F.A., 2013. Efficacy of moxidectin long-acting injectable formulation (1 mg/kg bodyweight) against first

98