Infection by Paramphistomidae trematodes in cattle from two agricultural regions in NW Uruguay and NW Spain

Accepted Manuscript Title: INFECTION BY Paramphistomidae TREMATODES IN CATTLE FROM TWO AGRICULTURAL REGIONS IN NW URUGUAY AND NW SPAIN Authors: J. Sanch´ ıs, R. S´ anchez-Andrade, M.I. Macchi, P. Pi˜ neiro, J.L. Su´ arez, C. Cazapal-Monteiro, G. Maldini, J.M. Venzal, A. Paz-Silva, M. Arias PII: S0304-4017(12)00408-6 DOI: doi:10.1016/j.vetpar.2012.07.028 Reference: VETPAR 6468 To appear in: Veterinary Parasitology Received date: 30-3-2012 Revised date: 24-7-2012 Accepted date: 30-7-2012 Please cite this article as: Sanch´ ıs, J., S´ anchez-Andrade, R., Macchi, M.I., Pi˜ neiro, P., Su´ arez, J.L., Cazapal-Monteiro, C., Maldini, G., Venzal, J.M., Paz-Silva, A., Arias, M., INFECTION BY Paramphistomidae TREMATODES IN CATTLE FROM TWO AGRICULTURAL REGIONS IN NW URUGUAY AND NW SPAIN, Veterinary Parasitology (2010), doi:10.1016/j.vetpar.2012.07.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Page 1 of 23 Accepted Manuscript 1 INFECTION BY Paramphistomidae TREMATODES IN CATTLE FROM TWO 1 AGRICULTURAL REGIONS IN NW URUGUAY AND NW SPAIN 2 J. Sanchís2, R. Sánchez-Andrade1, M.I. Macchi2, P. Piñeiro1, J.L. Suárez1, C. Cazapal-3 Monteiro1, G. Maldini2, J.M. Venzal2, A. Paz-Silva1,♦, M. Arias1. 4 1Epidemiology, Zoonoses and Parasitology (COPAR), Faculty of Veterinary, 5 University of Santiago de Compostela, 27002-Lugo (Spain). 2Parasitología, 6 Universidad de la República (Regional Norte), Salto (Uruguay). 7 8 ABSTRACT 9 The analysis of infection by Paramphistomidae trematodes was conducted in two 10 agricultural regions with different knowledge on this parasitosis. Faecal and blood 11 samples were collected from 374 cattle in Salto (NW Uruguay) where there is a lack of 12 information about paramphistomosis. A total of 429 cattle from Galicia (NW Spain), 13 an area with previous records of infection by gastric flukes, were sampled. Diagnostics 14 of trematodosis was developed by using a copromicroscopic probe and an ELISA with 15 excretory/secretory antigens collected from adult Calicophoron daubneyi 16 (Paramphistomidae) specimens. Results were evaluated according intrinsic and 17 extrinsic factors. 18 In the Uruguay, the percentage of cattle passing Paramphistomidae-eggs by faeces 19 was 7% (95% Confidence Interval 5, 10). A significantly higher prevalence of 20 paramphistomosis in the Hereford x Angus cattle (OR= 3.5) was recorded, as observed 21 for the oldest ruminants (>3.5 yr). An overall seroprevalence of 29% (25, 34) was 22 obtained by ELISA, with the highest values in the Friesians (OR= 3), the youngest 23 bovines (<2.5 yr) and dairy cattle (Friesians). 24 ♦ Correspondence: Dr. Adolfo Paz Silva. Tel: 982822116; Fax: 982252195; E-mail: [email protected] Page 2 of 23 Accepted Manuscript 2 Twenty-six percent (22, 30) of the cattle from Spain passed eggs by faeces, and cattle 25 aged 2.5-7 yr reached significant highest prevalences. By means of the ELISA, a 26 percentage of fifty-five cattle (50, 59) had antibodies against the gastric fluke, and the 27 highest seroprevalence was observed among the bovines under 6 yr. 28 It is concluded that paramphistomosis is on the increase in cattle from NW Spain, 29 partly due to the absence of an effective treatment against the trematode. There is a 30 need for reducing the risk of infection by Paramhistomidae spp in cattle from 31 Uruguay, especially by improving their management to avoid exposure to the gastric 32 trematode. Further studies are in progress for identifying the species of 33 Paramphistomidae affecting ruminants in Uruguay. 34 35 Keywords: Paramphistomidae, bovine, Uruguay, Spain, ELISA, risk factors 36 37 INTRODUCTION 38 Paramphistomosis is one of the most pathogenic diseases caused by gastrointestinal 39 trematodes in ruminants all over the world (Cringoli et al., 2004; Ilha et al., 2005; Díaz 40 et al., 2006; Kamaraj et al., 2010), responsible for heavy losses to the livestock 41 industry in subtropical and tropical areas (Khani et al., 2008). 42 The intermediate hosts are aquatic snails belonging to Lymnaeidae spp., living under 43 some particular conditions characterized by the presence of vegetation, humidity, 44 frequent rainfall and mild temperatures (Abrous et al., 1999; Arias et al., 2009; Pinedo 45 et al., 2010). As described for the trematode F. hepatica, the snails shed cercariae able 46 to swim actively and reach foliage, where transform into metacercariae, the infective 47 stages (Paraud et al., 2008). These are suitable conditions for the pasture can grow 48 throughout the year also. 49 Page 3 of 23 Accepted Manuscript 3 Infected animals are routinely detected by means of copromicroscopical techniques 50 (Tariq et al., 2008), but egg-counts can be influenced by some pathophysiological 51 changes in the definitive hosts, feed composition, the sampling hour or the amount of 52 faeces produced per day (Phiri et al., 2006). For trying to solve this situation, the 53 immunoenzymatic assay ELISA has been suggested, and higher values for sensitivity 54 and specificity when using the excretory/secretory antigens (89.4% and 87.2%, 55 respectively; Díaz et al., 2006) than the somatic products (23.65% and 85.71%, 56 respectively; Kaur et al., 2009) were achieved. 57 In the current investigation, two objectives have been planned. Firstly, to apply the 58 acquired experience on the diagnostics of bovine paramphistomosis in Spain for 59 gaining current information on this trematodosis in the Uruguay, due to only few data 60 are available (Cardozo et al., 1991). Secondly, to provide updated information from 61 Galicia (NW Spain), where cattle infected by Calicophoron daubneyi have been 62 detected six years ago (Diaz et al., 2006). 63 64 MATERIAL & METHODS 65 Areas of study 66 The current investigation comprises two agricultural regions, Salto (SW Uruguay) and 67 Galicia (NW Spain), where bovine farming represents the main economical input. 68 69 a) Salto (NW Uruguay) 70 Between September and December 2010, faecal and blood samples were collected 71 from 374 cattle belonging to 13 farms in the region of Salto (NW Uruguay) (31° 23' 72 00" S, 57° 58' 00" W). This is a 14,163 km² agricultural region with a cattle population 73 Page 4 of 23 Accepted Manuscript 4 about 820,000 animals focused to milk production (Friesian), meat (Hereford, Angus, 74 Hereford x Angus and Crossbred) or mixed/double aptitude (Normand). 75 As it is located in the temperate zone of the Southern hemisphere, the climate is 76 moderate with average temperatures ranging from 12ºC in the winter to 25ºC in the 77 summer. Significant amounts of precipitation occur in all seasons in most areas 78 (Kottek et al., 2006). 79 80 Cattle are often maintained under an extensive or semi-extensive regime feeding on 81 grassland, with water available from natural wells. There is observed rotation of 82 pastures according to the presence of herbage, and supplementation is provided when 83 necessary. Given that Friesians are moved to the milking room 1-2 times/day, water is 84 taken from potable drinkers. Cattle are usually dispatched at 8-9 yr. 85 Control of trematodosis is only focused against the infection by F. hepatica, consisting 86 of the administration of two dosages of nitroxynil to animals younger than 3 yr only. 87 88 b) Galicia (NW Spain) 89 From January to April 2010, a survey was conducted among 429 cattle in 49 farms 90 from NW Spain (42º20’–43º45’N, 6º49’–8º00’W). This is a 29,574 km² agricultural 91 area with a cattle population about 900,000 animals, where the numerically most 92 important breeds are Friesian (focused on milk production) and the autochthonous 93 Rubia Gallega (production of calves with outdoor grass fattening). 94 The climate in this area is oceanic, with presence of mild temperatures and frequent 95 rainfall throughout the year (Kottek et al., 2006; Díaz et al., 2007). 96 97 Page 5 of 23 Accepted Manuscript 5 Management conditions of the cattle in this region have been formerly summarized 98 (Arias et al., 2010). Briefly, Friesian cows are exploited under an intensive regime, and 99 receive fresh pasture or hay in relation to the season. Due to their dairy aptitude, 100 control of parasites is administered during the dry period, based on the application of 101 benzimidazoles (albendazole, triclabendazole) or macrocyclic lactones (eprinomectin). 102 Rubia Gallega cattle are maintained outside and are only brought into the paddocks at 103 night. Control of trematodes consists of the administration of albendazole against F. 104 hepatica in spring and autumn (Paz-Silva et al., 2007). 105 106 Sampling 107 Clinician veterinarians were responsible for the sampling in the two areas of study. 108 Faeces were obtained directly from the rectum in each cattle, and blood was 109 individually collected by tail vein punction. 110 Regarding the management of the ruminants in Salto (Uruguay), 3 age-groups were 111 considered, G-1 (<2.5 yr), G-2 (2.5 – 3.5 yr) and G-3 (>3.5 yr). Cattle younger than 3 112 yr old was considered as young while those above this age as adults. 113 Three groups were established according to the aptitude, dairy (Friesians), beef 114 (Hereford, Angus, Hereford x Angus and Crossbreds) and double or mixed aptitude 115 (Normands) (Table 1). 116 117 Cattle from Galicia (NW Spain) was divided into three groups regarding the age, G-1 118 (<2.5 yr), G-2 (2.5 – 7 yr) and G-3 (>7 yr). Two aptitudes were considered, dairy 119 (Friesian) and beef (Rubia Gallega). 120 121 Selection of the animals 122 Page 6 of 23 Accepted Manuscript 6 A list with all the bovine farms in each area was provided by a government quota 123 department, for which farmers are obliged to list all their cattle. For each cow, this 124 directory includes age, breed, aptitude, date of entry in the farm (purchase or birth), 125 and an identification number. The animals were randomly chosen by using numbers 126 changeably generated by a computer statistical package (SPSS for Windows, v. 18.1; 127 SPSS Inc., Chicago, IL, USA). 128 129 Diagnosis of infection by Paramphistomidae 130 a) Copromicroscopical analysis 131 Paramphistomidae-eggs were observed by means of the copromicroscopical 132 sedimentation technique, using individually samples of 5 g of faeces (Happich & 133 Boray, 1969). It should be remembered that C. daubneyi specimens have been 134 identified in cattle slaughtered in NW Spain (Díaz et al., 2006; Arias et al., 2011). 135 136 b) Enzyme Linked-Immunosorbent Assay (ELISA) 137 All the ELISAs were done in the COPAR Lab (Lugo, Spain). The humoral IgG 138 response was analyzed against C. daubneyi-excretory/secretory (CES) antigens 139 prepared from live adult flukes obtained from bovine slaughtered at a local abattoir 140 (Lugo, Spain), according to Paz-Silva et al. (2010). Briefly, paramphistomids collected 141 in the rumen and reticulum of infected animals were washed in PBS (phosphate 142 buffered saline, pH 7.4) and then incubated for 24 h at 37°C and 5% CO2 in RPMI 143 medium containing 0.5 mM PMSF. The medium was removed every 3 h, and then 144 centrifuged, dialyzed extensively against water and lyophilized. 145 ELISAs were performed in U-bottom microtiter plates (Costar®, Barcelona, Spain) 146 coated with 1 μg mL-1 CES, sera (tested in duplicate) at 1:40 and horseradish 147 Page 7 of 23 Accepted Manuscript 7 peroxidase conjugated rabbit anti-bovine IgG (H&L chains, Nordic Immunology 148 Laboratories, Tilburg, Netherlands) at 1:500. 149 Negative serum samples utilized as controls were obtained from 37 3-month calves 150 remaining stabled from their birth and negative to coprology during this period. 151 The cut-off value was estimated as the mean of absorbances from the negative controls 152 plus three standard deviations (Sánchez-Andrade et al., 2001). Thus, positive 153 absorbance values were higher than 0.3430. 154 155 c) Sensitivity and specificity 156 The estimation of sensitivity and specificity was done by using sera from 83 cattle 157 slaughtered at a local abattoir, where the digestive tract (oesophagus, rumen and 158 reticulum) of each animal was checked for the presence of gastric flukes. 159 160 d) Analysis of the cross-immune reaction 161 The analysis of cross-immunity was conducted by using sera from cattle monoinfected 162 by Paramphistomidae or F. hepatica. Infection was assessed by the simultaneous 163 demonstration of eggs in the faeces and serum antibodies against the respective 164 excretory/secretory antigens (Arias et al., 2009). 165 Accordingly to this, the sera of 103 cattle with paramphistomosis were faced to F. 166 hepatica excretory/secretory antigens. Similarly, serum samples from 94 cattle 167 infected by F. hepatica were tested against CES. 168 With the aim to corroborate the cattle were monoinfected, two samples were taken for 169 each animal, at a 4-month interval. 170 171 Statistical analysis 172 Page 8 of 23 Accepted Manuscript 8 The percentages of prevalence have been expressed as the value and the 95% 173 confidence interval, and these were analyzed by using the χ2 test. Differences were 174 considered as significant when P < 0.05. 175 By taking into account the values of sensitivity and specificity provided in previous 176 investigations (89% and 87%, respectively; Paz-Silva et al., 2010), the prevalence was 177 estimated as the true prevalence according to the following expression: 178 179 True prevalence = (AP + SP-1) / (SP + SE -1) 180 181 where AP means the apparent prevalence, SP the sensitivity and SE the sensibility. 182 Results were analyzed regarding the breed, age and aptitude of the animals. By 183 considering that cattle from NW Spain belonged to two different breeds with other 184 different aptitude, Friesian (dairy) and the autochthonous Rubia Gallega (beef), the 185 analysis of the influence of aptitude was not carried out. 186 187 The possible relationship between the breed, age and the prevalence of 188 paramphistomosis was established by calculating the odds ratio (OR) values 189 (Thrusfield, 2005; Arias et al., 2011). All the data were classified by using the chi-190 squared automatic Interaction detector (CHAID) test (Arias et al., 2010). This is often 191 used as an exploratory technique and is an alternative to multiple linear regression and 192 logistic regression. CHAID is used to build a predictive model, based on a 193 classification system (Magidson, 1994). The analysis subdivides the sample into a 194 series of subgroups that share similar characteristics towards a specific response 195 variable and that maximises the ability to predict the values of the response variable. 196 The first predictor category (on which the sample will be split) is the predictor that is 197 Page 15 of 23 Accepted Manuscript 15 Another explanation could be that weak infections mean a limited stimulus to the 345 immune system, although further studies are needed for confirming it. 346 347 CONCLUSIONS 348 Regardless bovine paramphistomosis has been detected some years ago in NW Spain, 349 the prevalence is increasing possibly due to a misdiagnosis and consequently the 350 application of ineffective treatments. Our results reflect a moderate geographical 351 distribution of paramphistomosis in this region, where a notable contamination of 352 grasslands seems responsible for almost half of the cattle exposed to 353 Paramphistomidae develop a patent infection characterized by the presence of egg-354 output (adult stages). 355 Although there is a lack of previous studies in Uruguagy, the current investigation 356 demonstrated a low to medium risk of infection in cattle possibly due to a low 357 percentage (7%) of bovines are contaminating pastures by shedding eggs. The elevated 358 percentage of farms in Uruguay with seropositive cattle led us to conclude that 359 paramphistomosis appears widely distributed. Further studies are in progress for the 360 identification of the Paramphistomidae species affecting cattle in Uruguay. 361 362 CONFLICT OF INTEREST 363 All authors declare the absence of any financial or personal interests that could 364 inappropriately influence the current work. The final article has been approved by all 365 authors. The ELISAs used in this study were developed at the University of Santiago 366 de Compostela (Spain). 367 368 ACKNOWLEDGEMENTS 369 Page 16 of 23 Accepted Manuscript 16 This research has been supported in part by the Project XUGA 370 PGIDT06RAG26102PR (Xunta de Galicia, Spain). Jaime Sanchís was given a 371 research grant (Edmundus 17; Banco de Santander – USC, Spain) for developing his 372 PhD Thesis at the University of Santiago de Compostela (Spain). 373 374 REFERENCES 375 Abrous, M., Rondelaud, D., Dreyfuss, G., 1999. Paramphistomum daubneyi and 376 Fasciola hepatica: influence of temperature changes on the shedding of 377 cercariae from dually infected Lymnaea truncatula. Parasitol. Res. 85, 765-378 769. 379 Arias, M., Lomba, C., Dacal, V., Vázquez, L., Pedreira, J., Francisco, I., Piñeiro, P., 380 Cazapal-Monteiro, C., Suárez, J.L., Díez-Baños, P., Morrondo, P., Sánchez-381 Andrade, R., Paz-Silva, A., 2011. 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Helminthol. 82, 373-481 376. 482 483 Page 21 of 23 Accepted Manuscript 21 Table 1.- Analysis of infection by Paramphistomidae spp. in cattle from Uruguay (South 483 America) by coprological and immunoenzymatic probes (N= 374). 484 485 Cattle Coprology CES-ELISA N Positive (Nr) Prevalence 95% CI OR Positive (Nr) True Prevalence 95% C I n gus 58 3 5 (0, 11) 16 28 (18, 40 ) o ssbred 61 4 7 (0, 13) 15 25 (15, 37 ) i esian 92 4 4 (0, 9) 42 46 (36, 56 ) r d x Angus 16 3 19 (0, 38) 6 38 (19, 61 ) r eford 100 12 12 (6, 18) 29 29 (21, 38 ) rm and 47 1 2 (0, 6) 2 4 (0, 14) Statistics (χ2= 9.943, P= 0.077) (χ2= 27.303, P= 0.001) < 2.5 47 2 4 (0, 10) 0.5 18 38 (26, 53 ) – 3.5 128 4 3 (0, 6) 0.3 25 19 (14, 27 ) > 3.5 199 21 11 (6, 15) 3.3 67 34 (27, 40 ) a tistics (χ2= 7.121, P= 0.028) (χ2= 9.543, P= 0.008) B eef 235 22 9 (6, 13) 66 25 (20, 31 ) D airy 92 4 4 (0, 9) 42 46 (36, 56 ) M ixed 47 1 2 (0, 6) 2 4 (1, 14) a tistics (χ2= 4.562, P= 0.102) (χ2= 26.213, P= 0.001) 486 487 Page 22 of 23 Accepted Manuscript 22 Table 2.- Analysis of infection by Paramphistomidae spp. in cattle from Galicia (SW Europe) 487 by coprological and immunoenzymatic probes (N= 429). 488 489 Cattle Coprology CES-ELISA N Positive (Nr) Prevalence 95% CI OR Positive (Nr) True Prevalence 95% CI r iesian 209 50 24 (18, 30) 116 56 (49, 62) i a Gallega 220 62 28 (22, 34) 119 54 (48, 61) Statistics (χ2= 1.007, P= 0.316) (χ2= 0.086, P= 0.769) < 2.5 33 5 15 (3, 27) 0.5 7 21 (11, 38) 2 .5 – 7 181 79 44 (36, 51) 5.1 132 73 (66, 79) > 7 215 28 13 (9, 18) 0.2 96 45 (38, 51) t atistics (χ2= 49.995, P= 0.001) (χ2= 47.980, P= 0.001) 490 491 Page 23 of 23 Accepted Manuscript 23 491 Table 3.- Combined analysis of bovine paramphistomosis among cattle from Uruguay (South 492 America) and Spain (SW Europe) by means of coprological and immunoenzymatic probes. 493 494 Salto (SW Uruguay) Galicia (NW Spain) Sedimentation ELISA N % 95% CI N % 95% CI + + 12 3 (1, 5) 106 25 (21, 29) + - 15 4 (2, 6) 6 1 (0, 3) - + 98 26 (20, 29) 129 30 (26, 34) - - 249 67 (64, 73) 188 44 (39, 49) 374 429 495 496 497 498