Epidemiology of Oestrus ovis (Linneo, 1761) infestation in goats in Spain

Veterinary Parasitology 130 (2005) 277–284 www.elsevier.com/locate/vetpar

Epidemiology of Oestrus ovis (Linneo, 1761) infestation in goats in Spain M. Alcaide *, D. Reina, E. Frontera, I. Navarrete Parasitology Section, Department of Medicine and Animal Health, Faculty of Veterinary Sciences, University of Extremadura, 10071 Ca´ceres, Spain Received 7 December 2004; received in revised form 21 February 2005; accepted 16 March 2005

Abstract This survey was conducted to determine the chronobiology and seroprevalence of nasal bot infestation (Oestrus ovis) in Spain and to identify the risk factors associated with this disease in caprine herds. A total of 1590 sera from adult goats were collected at random on 175 farms in southwestern Spain. Sera were tested by ELISA, using crude protein from second stage larvae as antigen. The mean seroprevalence was 46.04% and mean percentage of optical densities was 41.83. These data indicate a high prevalence of this parasite in the investigated areas. The serological survey revealed that goats managed at higher altitudes, at meridians latitudes and on farms with small herds had a smaller probability of infestation. Eighty goat heads, obtained from abattoirs in the central region of Spain, were collected and examined for nasal botflies from February to October 2002. O. ovis larval stages were recovered from the nasal-sinus cavities of 23 goats, reaching a prevalence of 34.94%. The mean larval burden was 3.9 larvae per infested head. No first instars were found during February and March, when the second instar reached its larger count. The third instar was observed in very small number during the whole period of study, with one peak occurring in July–August. These data show the existence of a favourable period for the development of larval instars of O. ovis in goats that starts in February and finishes in September. # 2005 Elsevier B.V. All rights reserved. Keywords: Oestrus ovis; Goat; Prevalence; Risk factors; Spain

1. Introduction Oestrus ovis (Diptera: Oestridae) is a cosmopolitan parasite that affects small ruminant livestock. The pathogenic effects are particularly serious in sheep and goats, on the larvae develop in the sinu-nasal passages. * Corresponding author. Tel.: +34 927 257 130; fax: +34 927 257 110. E-mail address: [email protected] (M. Alcaide).

The O. ovis larvae elicit clinical sings of cavitary myasis as a seromucous or purulent nasal discharge, frequent sneezing, incoordination and dyspnea. This infection can resulting signs of generalised disease, causing serious economic losses in sheep and goat herds reared for meat and dairy produce. O. ovis can thrive in different environments (Horak, 1977; Breev et al., 1980; Pandey and Ouhelli, 1984) and can overwinter in a diapause state, as larvae in the sinu-nasal passages of the host (Cobbett and Mitchell,

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1941; Horak, 1981). The ability to adapt to different environments allows to the natural persistence of infestation and the difficulties for its control. Recent surveys have demonstrated the high prevalence of caprine oestrosis in numerous areas all over the world, e.g. 48.3% in India (Jagannath et al., 1989), 31.3% in Mexico (Martı´nez et al., 1992), 4% in Argentina (Trezeguet, 1996) and 53.8% in Nigeria (Biu and Nwosu, 1999). In Mediterranean countries, the prevalence described was 91% in Greece (Papadopoulos et al., 1997), 28.4% in France (Dorchies et al., 2000) and 14% in Morocco (Berrag et al., 1996). Despite the importance of parasitism by O. ovis in small ruminants and the high number of goats breeding in Spain, the knowledge of the chronobiology and prevalence of this myiasis in caprine livestock never have been recorded. This study attempted to determine the seroprevalence of O. ovis infestation in Spain and the influence of potential risk factors associated with this myiasis in goats herds, the seasonal variations in the life cycle of O. ovis and the association between the presence of larvae and weather conditions (temperature and rainfall).

2. Materials and methods 2.1. Animals A total of 80 adult goats from farms located in the central provinces of Spain (Ca´ ceres, Ciudad Real and Toledo) were examined from February to October 2002 for O. ovis larvae infestation. Data on the origin, sex and age were recorded from each animal. The heads of slaughtered goats were separated from the bodies, incised sagitally using an electric saw and examined for O. ovis larvae, in the nasal-sinus cavities, according to previously described methods (Yilma and Dorchies, 1991). The larvae found in the nasal and sinus cavities were recovered, counted and identified according to keys described by Zumpt (1965). 2.2. Sera The serum samples were obtained from the Animal Health Regional Laboratories in different provinces of

the southwestern Spain (Badajoz, Ca´ ceres and Sevilla). A total of 1590 sera from adult goats (>6 months of age) were examined. They were routinely collected for the brucellosis surveillance program in these regions between November 2000 and September 2002. Data regarding location, number of animals and approximate age were recorded for each checked farm. According to the official census, in December 1999, the total number of goats in Badajoz was 89,909, in Ca´ ceres 154,021 and in Sevilla 173,895 (Fig. 1). The number of animals investigated represented approximately 0.44% in Badajoz (n = 395), 0.51% in Ca´ ceres (n = 783) and 0.24% in Sevilla (n = 412) concerning the total caprine population of each province. A total of 175 farms were surveyed, 45 from Badajoz, 74 from Ca´ ceres and 56 from Sevilla. 2.3. Study area The study was conducted in a Spanish area located between the 39.3th and 36.6th north parallel and the 7th and 4th west meridian (Fig. 1). The weather is characterised by variable rainfall, occurring mainly in spring and autumn and usually does not exceed 100 mm per month during these periods. Winter is mild and dry with a mean temperature around 8.3 8C and rainfall of 84.5 mm per month. Summer is long, hot and extremely dry, with average temperature and rainfall of 25 8C and 19.5 mm per month (Table 2). The majority of the herds studied were maintained primarily in cultivated lands, essentially dry farming and mountain pastures, no more than 1100 m above sea level, while the remainder were on the grazing lands of traditional Mediterranean forest. 2.4. Enzyme-linked immunosorbent assay (ELISA) Sera samples were obtained and stored at
20 8C until use. The ELISA method used was previously described by Dombre (2000). Second instars were obtained from heads of naturally infected goats. After the larvae were homogenised, the extract was centrifuged at 450  g for 20 min at 4 8C, the supernatant removed and its protein concentration determined by a modified method of Bradford (Coomasie Plus Protein Assay, Pierce; Ref1856210). Plates were incubated with the antigen at

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Fig. 1. Map of Spain showing the location of the investigated provinces and theirs official veterinary caprine census in 2000: (a) Ca´ ceres: 154,021 goats, (b) Badajoz: 89,909 goats, (c) Sevilla: 173,895 goats, (d) Ciudad Real: 133,000 goats and (e) Toledo: 68,000 goats.

5 mg/ml for 3 h at 37 8C. After washing, PBS 5% skimmed milk solution was added for blocking the wells. After triple washings, 100 ml of duplicated sera diluted at 1:200 in PBS with 0.1% Tween 20 were added and newly incubated at 37 8C for 45 min. After washing, 100 ml of donkey anti-goat IgG (whole molecule) peroxidase conjugate (Sigma, A-5420) diluted at 1:5000 was added per well. Finally, 100 ml of substrate solution with citrate buffer 0.05 M (pH 5.0), hydrogen peroxide and 3,30 ,5,50 tetramethylbencidine (Sigma, T-3405), was incubated for 20 min and stopped with 3N sulphuric acid. Positive controls were evaluated by repeated titration of several serum samples from naturally infested animals from the studied regions. Serum samples from young treated goat, kept indoors and without possible contact with adult bot flies, served as negative controls. The cut-off value (0.4) for positive was defined as herring two standard deviations ( p < 0.05) above the mean optical density for negative control serum.

2.5. Data analysis The percentage of antibodies (mean percentage of OD) was calculated for each sera sample by comparison with positive and negative reference sera as follows: Percentage of antibodies ODðserum sampleÞ
ODðnegative control seraÞ ¼  100 ODðpositive control seraÞ
ODðnegative control seraÞ

The seroprevalence data were calculated as: Percentage of seroprevalence total number of serum samples with OD > 0:4 ¼  100 total number of goats examined

Quantitative variables (seroprevalence and percentage of antibodies) were categorised according to the potential risk factors (latitude, altitude, flock size and caprine population density). Statistical comparisons were evaluated by ANOVA, in order to investigate the

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significant differences among groups. Pearson correlation was estimated between seroprevalence and percentage of antibodies in relation to the potential risk factors. The monthly mean burden of the different larval instars was estimated as a percentage of the total larvae collected per month. Statistical comparisons were evaluated by the Kruskal–Wallis test, in order to investigate monthly variations. Pearson correlation was estimated between seasonal variations, burden and climatic conditions (temperature and rainfall). Data analyses were performed using a commercial statistical package, SPSS 11.0 program for Windows (Microsoft).

3. Results

Fig. 3. Within-flock seroprevalence of O. ovis infestation and the normal distribution estimated in caprine livestock from southwestern Spain.

3.1. Seroprevalence and percentage of antibodies The results of the seroepidemiological survey showed that 717 animals were seropositive among the 1590 goats analysed. The estimated seroprevalence was 46.04  30.28% and the percentage of antibodies was 41.83  23.89%. When analysed by province, the seroprevalence was 52.12% in Badajoz, 37.43% in Ca´ ceres and 52.75% in Sevilla, while the percentage of antibodies ranged between 47.91% in Badajoz and 33.75% in Ca´ ceres (Fig. 2). These differences were significant between Ca´ ceres relative to Badajoz and Sevilla. The detection of anti-Oestrus antibodies was significantly associated with the seroprevalence (r = 0.89; p < 0.000). By farms, 91.43% (n = 160) of sampled herds had seropositive goats. Only 15 farms were found to be

Fig. 2. Mean percentage of antibodies and seroprevalence of Oestrus ovis infestation by provinces of goats from southwestern Spain.

free of infested animals, while a total of 14 had all their animals seropositive (Fig. 3). The normal distribution calculated for the investigated caprine population was around the average (46  30.3%). 3.2. Potential risk factors There was a negative but not significant correlation between seroprevalence and percentage of antibodies concerning latitude and altitude (r =
0.1; p = 0.2 and r =
0.07; p = 0.3). In fact, the lowest number of seropositive animals was found in locations over 38.88N parallel and higher altitudes, more than 650 m above sea (Table 1). A positive and significant correlation (r = 0.26; p < 0.000) was found between flock size and seroprevalence and percentage of antibodies. So, very large herds (more than 400 goats), large (between 75 and 400 goats) and medium ones (between 30 and 75 goats), were more likely to have seropositive goats and higher percentages of antibodies than small herds (less than 30 goats) (Table 1). There was also a significant positive correlation between caprine population density and seroprevalence, as well as concerning percentage of antibodies (r = 0.23 and r = 0.22; p < 0.005). Hence, in regions with 5–7.5 goats/km2 there was a less probability of finding seropositive animals than in more populated areas (Table 1).

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Table 1 Seroprevalence and percentage of antibodies against Oestrus ovis in goats from southwestern Spain according to the risk factors studied Number of goats examined

Number of herds examined

Seroprevalencea (%)

Percentage of antibodiesa

Latitude 40.2–39.58N 39.5–38.88N 38.8–38.18N 38.1–37.48N 37.4–36.78N

363 352 349 141 385

40 35 36 16 46

48.71 28.37 55.41 55.34 55.41

b a b b b

42.69 26.28 50.74 53.16 43.79

b a b b b

Altitude (m) <250 250–450 450–650 >650

248 669 406 267

46 59 41 27

52.06 43.61 55.78 26.28

b b b a

46.76 40.96 47.28 27.09

b b b a

Herd size (goats) <30 30–75 75–400 >400

425 595 296 274

58 47 44 24

22.19 56.62 56.36 67.73

a b b b

26.12 47.91 44.99 60.16

a b b c

40 42 32 59

47.83 26.77 55.20 53.58

b a b b

45.28 25.19 46.31 48.92

b a b b

Risk factors

Caprine population density (goats/km2) <5 400 5–7.5 349 7.5–10 390 >10 452 a

Values with different letters (a, b, c) for a risk factor in the same column are significantly different ( p < 0.05).

3.3. Prevalence and intensity The number of goats infested with O. ovis larval stages was 23 (34.94%). Only two herds were free of infested animals, reaching a percentage of 84.62%. The monthly incidence of infestation with O. ovis and

the analysis of the percentage of each larval stage in relation to the total larval burden are shown in Table 2. July had the highest rate of infestation (40.0%). Even there was no significant difference between the monthly prevalence during the study. The mean intensity of infestation was 3.91 larvae by infested

Table 2 Monthly results concerning examined heads, incidence of infested animals, mean larval burden per head, percentages of first, second and third larval stage (L1–L3), respecting the total larvae found per month in goats from the central region of Spain, temperatures and rainfall in the studied regions Year

Month

Number of heads examined

Incidence (%)

Mean larval burden

L1 (%)

L2 (%)

L3 (%)

Temperature (8C)

Rainfall (mm)

2002

February March April May June July August September October

5 5 9 5 6 10 10 10 20

20.0 20.0 22.2 20.0 33.3 40.0 30.0 20.0 35.0

4 1 4 2 3.5 2.75 4 5.5 8.43

0.0 0.0 12.5 50.0 71.4 45.5 92.3 100.0 98.3

100.0 100.0 87.5 50.0 28.6 45.5 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 9.0 7.0 0.0 1.7

10.3 12.2 14.2 16.7 23.6 25.8 24.8 20.9 17.9

10.6 97.2 53.6 17.9 1.6 0.0 6.6 41.9 102.9

80

34.94

3.91

1.97

18.5

36.9

Total

52.22

45.73

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head. The highest mean larval burden was found during autumn, in October 8.43 larvae were found per infested head indeed, the highest number of larvae recovered from a single goat (45 larvae) was observed in October. The mean percentage of L1 was the 52.22, while L2 and L3 represented 45.73 and 1.97, respectively, of the total larval burden. 3.4. Seasonal evolution according to climatic conditions Significant differences were found between the percentages of the larval stages during the study ( p < 0.01). No L1 were found during February and March. The first L1 were recovered in April, with a progressive increase until reaching the first peak in June. After that, the percentage of L1 decreased during July, but peaked again to a greater level of this percentage during August through October (Table 2). The percentage of L1 was positively correlated with monthly rainfall (r = 0.714; p = 0.03), while no significant negative correlation with temperature was observed (r =
0.058; p = 0.8). The percentage of L2 showed a significant rise from February to April. After this, the percentage of L2 began to decrease, describing some fluctuations, until it reached its lowest level in August. The lowest percentage of L2 coincided with the highest percentage of L1 and vice versa, because of the lack of influence caused by the very low percentage of L3 in relation to the total maggots recovered, there was only one peak observed during July–August (Table 2). The correlation between the percentage of L2 and the climatic conditions was negative and significant for the temperature (r =
0.76; p = 0.01), but not significant for rainfall (r =
0.09; p = 0.82). In contrast, a significant positive correlation was found between the percentage of L3 and the average temperature (r = 0.69; p = 0.03) but a negative correlation for the monthly rainfall (r =
0.48; p = 0.19).

4. Discussion and conclusion Due to the lack of knowledge on how widespread is the caprine oestrosis and its severe effects in livestock reported by farmers in Spain, this large-scale (n = 1590) seroepidemiologic study was conducted.

The results demonstrated that O. ovis is a common parasite among herds examined, with a seroprevalence of 91.43%. The infestation was widespread on the caprine farms, with a high seroprevalence of 46.04% and a mean percentage of optical densities of 41.83%. The estimated overall seroprevalence for the Spanish caprine population has been found greater than the values obtained from other Mediterranean countries, such as France (Dorchies et al., 2000) and Morocco (Berrag et al., 1996). The relationship of prevalence according to latitude was previously observed by Ruiz-Martı´nez et al. (1992) who described differences in the annual chronology of the cycles in geographical points with different latitudes, specifically Spain and Morocco. Our study reported that goats from meridian locations, of Badajoz and Sevilla, were more likely to have higher prevalences than goats from northern locations, such as Ca´ ceres (Fig. 2). The relationship between the prevalence of infestation and altitude, resulted in a decreased number of seropositive goats of farms situated over 650 m above level sea (Table 1). It may be due to the weather conditions at these altitudes which are not favourable for the presence and development of O. ovis, as described Bergeaud et al. (1994). The number of seropositive goats correlated with flock size, as many other authors (Gupta et al., 1985; Marchenko and Marchenko, 1989; Ribeiro et al., 1990) reported there was no association between prevalence of infestation and flock size. Although Bauer et al. (2002) and Murguı´a et al. (2000) confirmed this association. In our study have been observed that small herds had significantly fewer parasitized animals than the large ones. This is possibly due to higher risk of infestation and reinfestation among herds of greater number of susceptible goats that are crowed together. CepedaPalacios and Scholl (2000) observed that the number of strikes by O. ovis gravid females increased in proportion to the number of goats gathered together grazing, specifically, there were a greater number of strikes in large herds than in smaller ones. Furthermore, the significant positive correlation between seroprevalence levels and caprine population density obtained, confirmed a higher risk of infestation in herds with more animals. The results of the seasonal variations in the larval burden distribution of O. ovis in goats in the central

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region of Spain, reaffirm that is a very common parasite of the goat herds checked (84.62%) and widespread within the goat population (34.94% of prevalence). The mean infestation intensity was 3.91 larvae per host, much lower than results obtained by Alcaide et al. (2003) in the sheep of southwestern Spain with a mean larval burden of 18.54 per animal. According with these results, Jagannath et al. (1989) and Dorchies et al. (2000) demonstrated under field observations, that the larval burdens were higher in sheep than in goats. The highest peak of L1 during this survey was registered in September and October that may be due to larvae deposited by gravid female adults in the nostrils of goats and the deceleration of the larval development during these months. The percentage of L2 showed a significant rise in February and April, and then began a decline, reaching their lowest level in August. The third larval stage was practically absent during the study, except in summer months. These results demonstrated that the survival of L1 is less in goats than has previously been described in sheep (Alcaide et al., 2003), as very few larvae can achieve development to L3 in goats. These differences in larval development between sheep and goats may be due to the ability showed by goats to avoid the larvae laying by adult flies and poor parasite–host compatibility that results in a severe local immunity reaction, with an important inflammatory cell infiltration against the larvae in the sinu-nasal cavities as reported by Dorchies et al. (1998) and Nguyen et al. (1999). O. ovis larvae have the capacity to adjust their developmental cycle during the parasitic phase, depending on the particular climatic conditions. When environmental conditions are adverse, larvae cease their development during the hypobiotic period (Cobbett and Mitchell, 1941; Horak, 1981). It was not possible to confirm the existence of a real hypobiotic period of L1 in Spanish goats, due to the absence of data on the larval distribution during the coldest months. In spite of that, there was a long time for the development of larval instars, L2 and L3 were recovered from February until September. Compared with ovine surveys in other Mediterranean regions, such as Sardania (Scala et al., 2001), Tunisia (Kilani et al., 1986), Sicily (Caracappa et al., 2000) and even, Spain (Alcaide et al., 2003), all of them reported the

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existence of a long favourable developmental period practically the entire year. In conclusion, O. ovis is very commonly found and widely disseminated among caprine herds in Spain. The favorable period for the development of the larval instars in goats starts in February and finishes in September. These findings confirm that there is a very high risk of infestation and reinfestation for susceptible animals located in meridian latitudes, low altitudes, large flock size and high caprine population density. Control of caprine oestrosis is recommended to prevent the economic effects of this goat parasitism in Spain.

Acknowledgements The authors wish to express their gratitude to the personal from the Animal Health Regional Laboratories from Badajoz, Ca´ ceres, Co´ rdoba and Sevilla and the colleagues from Veterinary Faculty of Co´ rdoba and to the slaughterhouses veterinarians and the rest of staff, from Coria and Ca´ ceres for their indispensable help in the sampling process.

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