THE STUDY OF ECTOPARASITISM WITH DERMANYSSUS GALLINAE IN BIRDS: ECOBIOLOGY, PATHOGENESIS AND CONTROL

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1 UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE CLUJ-NAPOCA PHD SCHOOL FACULTY OF VETERINARY MEDICINE CRISTIAN ALEXANDRU MAGDAŞ THE STUDY OF ECTOPARASITISM WITH DERMANYSSUS GALLINAE IN BIRDS: ECOBIOLOGY, PATHOGENESIS AND CONTROL Summary of PhD thesis SCIENTIFIC COORDINATOR Prof. Dr. D.H.C. ERONIM ŞUTEU CLUJ-NAPOCA

2 THE STUDY OF ECTOPARASITISM WITH DERMANYSSUS GALLINAE IN BIRDS: ECOBIOLOGY, PATHOGENESIS AND CONTROL SUMMARY D. gallinae affects especially chickens, reason why, it is known in the entire world as chicken red mite. In some European countries, D. gallinae is the most important ectoparasite of the layers, the damages that this parasite makes, in european poultry husbandry are estimated at approximately 130 milion euros every year, through the reduction of egg production, the formation of eggs with lower weight or with modified shell and moreover through mortality that can reach 50 %. The incidence of this parasitic disease is higher in small households, but it can also have serious consequences in intensive exploitation units. This parasitic disease was reported in over 30 species of domestic and wild birds, is exceptional in horses, cows, rabbits, dogs, rats and including humans. It has a great importance as a transmitter and vector of some viral and bacterial pathogens. Because of the growing importance of the infestation with mites on birds, in the last years, there were discovered approximately 35 molecules of acaricides that can be used in mite infestation. However, their numbers is restrained by the legislation in some cases and by the appareance of chimioresistance for some molecules. On international level, aspects regarding chemical pharmaceutical polution are being studied more and more, through the accumulation of active potential waste (mutagen, cancerigen, teratogen) in products from animals under antiparasitic treatment. The way to avoid this drawbacks could be the use of pharmaceutical products of organic nature from the vegetal reign. In veterinary practice, the aplication of fitotherapy offers the developement of therapeutic treatment procedures and in combination with other approaches, it offers a higher level of safety in therapy, treating more carefully humans, animals and the environment. It s obvious that, in the fight against parasitic diseases, the judicious junction of more methods must strongly be considered, and that is why the usage of only few of the methods can t be fully effective. 349

3 1. RESEARCHES CONCERNING THE REPRODUCTION OF THE EXPERIMENTAL INFESTATION WITH DERMANYSSUS GALLINAE IN CHICKENS In the first chapter of our research, we wanted to perform the experimental reproduction of the infestation with D. gallinae in broiler chickens and the evaluation of the pathogenic effects, through the definition of some haematological, biochemical, fluctuating weight parameters and observe the clinical and anatomopathological changes. The researches were performed during October-November 2005, on 20 broiler chickens with the age between 4 and 7 weeks, separated in two groups of 10 chickens. The two groups of chickens were placed at different locations to avoid any posibility of contamination of the control group and they benefited by the same environmental and biotope conditions. Ectoparasites were gathered from a laying hens farm form Cluj county, where the infestation with D. gallinae was identified. Before the infestation, the groups were weighed and then for a period of 3 weeks, the measurements were performed once a week and the average daily growth was calculated. To highlight the anaemic, hemolytic, toxic and spoliator effects of the ectoparasites there were 2 haematological and biochemical determinations conducted, before the infestation (day 0) and after the infestation (day 21) respectively. Through the pierce of the axillary vein was taken blood on anticlothing agent EDTA from every chicken individualy. Serum was taken for the biochemical and enzyme tests. We performed the following haematological determinations: erythrocyte count, haemoglobin dosage, hematocrit determination. We performed the following biochemical determinations: the blood urea, the triglycerides, the glucose, the γ-glutamyltransferase (GGT), the alkaline phosphatase (ALP). To highlight the morphopathological aspects, the birds were sacrificed on the 21 day of the experiment and the anatomopathological examination was performed. The statistic data calculation was done in Windows XP operating system, Statistix 9 program. For every group and every blood taking the following statistical indices 350

4 were established: the standard average and deviation, also the T test for comparing the means for 2 pair groups. Results In day one of the experiment, the body weight of the chickens from the infested lot with D. gallinae recorded an average of 2285±123,22 g and the one from the control group registered an average of 2600±158,271 g, with a difference of 315 g between the medium weight of the chickens from the 2 groups A significant difference between the weight of the 2 chicken lots was disclosed at the end of the experiment (p=0,004). The clinical exam that was made at the end of the experiment revealed normal aspects of the chicken from the control group and paleness of the comb, wattles and outward mucous membranes of the chickens from the infested lot. After 3 weeks after infestation, in the infested group, the values for the red blood cells ( ±172000/mm 3 ), haematocrit (25,2±1,316%) and the ones for haemoglobin (8,04±0,627 g/dl) were lower than the initial values and than the control group values with significant distinct statistic differences. After calculating the medium parameters of the red blood cells, anaemia was hipochromic and macrocytic in the case of the lot infested in day 21 of the experiment. Glycemia of the infested group was 172,4±11,711 mg/dl after the infestation and the one of the control group was 178,6±15,993 mg/dl. At both groups, the medium values of the glycemia, were similar, at the beginning and at the end of the experiment, being situated in the normal limits of the species. There were some very significant/statistic significant differences of the groups between the values of glycemia registered at the beginning of the experiment and the one from the end of it, but there weren t any statistic significant differences revealed between the infested lot and the control group at the beginning of the experiment (p=0,853) either at the end (p=0,425). The triglycerides were lower in the case of the infested group, with a difference of 56±11,401 mg/dl, from the initial value of 100,3±21,291 mg/dl, having lower values than the control group (89±5,099 mg/dl) in day 21 of the experiment and the differences being very significant (p=0,0002 ; p=0,002). 351

5 At the end of the experiment, the values of the blood nitrogen were higher than the initial ones for both lots with an average of 20,33±3,428 mg/dl for the infested group and 17,68±9,957 mg/dl for the control group. There were some statistic significant differences in case of both lots between the values of the urea at the beginning and at the end of the experiment, but there weren t any statistic significant differences between the recorded values of the urea from the 2 group of chickens in day 21 of the experiment. After infestation, the ALP values grew at both groups of chicken, with an average of 737,1±622,58 U/l ( U/l) for the infested lot and with a media of 925,4±674,255 U/l ( ) for the control group A, significant difference between the ALP values from the beginning and from the end of the experiment was revealed in the case of the infested group, but the difference between the control group and the infested group at the end of the experiment was statistically insignificant (p=0,581). The GGT values were - after the infestation, situated between 22,1 and 43,4 U/l in the case of the infested group, and for the control group the GGT values were between 27,7 and 33,8 U/l, with an average of 31,96±2,481 U/l. It was the same situation as the one for the values of ALP: a statistic significant difference between the GGT values registered at the beginning and at the end of the experiment for the infested lot (p=0,03) and a statistic insignificant difference between the control group and the infested group, at the end of the experiment. (p=0,854). The morphopathological exam made at the end of the experiment revealed modifications in the case of the infested group. Macroscopically, there was an anaemic aspect of the carcass and a necrotic milliary hepatitis. Histopathologically, degenerative lipidic and necrotic lesions, situated especially near the centrolobular vein, were revealed in the centre of contagion. 2. STUDY ON THE EFFECT OF THE INFESTATION WITH DERMANYSSUS GALLINAE ON THE PRODUCTION OF EGGS AND ON THE IMMUNITY OF HENS The objectives for this chapter are: 352

6 A. The determination of some hematological parameters (total red blood cells, hemoglobin, haematocrit) on battery hens that were infected with D. gallinae and on the ones that were not. B. The determination of the influence the infestation with D. gallinae has on the egg laying. C. The influence of the infestation with D. gallinae on the immune response mediated on a cellular and humoral level for egg laying hens. The research was carried out during August-September egg laying hens with the age of 36 weeks were studied and divided into the following categories: - group I 20 battery hens infested with D. gallinae and coming from SDE Floreşti; - group II 20 battery hens, treated against external parasites, from the FMV Biobase Cluj Napoca which represent the negative witness lot. All the birds that were studied provened from the same poultry farm. The two hen groups benefited from de same conditions during the experiment (they were kept in battery boxes, fed and received the same light program). Both groups received antiparasitic - albendazole 15mg/kc, one administration. Group II was received a product for external parasites - Ectopor, 0.1 ml/kc, spot-on application and was maintained for 30 days before the experiment started in conditions that didn t allow the infection with D. gallinae. The experimental protocol followed the next aspects: - day 0 of the experiment: blood collecting for the determination of some hematological parameters (total red blood cells, hemoglobin, haematocrit) and for the procurement of serum; inoculation of 0.5 ml suspension 5% sheep red cells, s.c. in the lateral thoracic region; the performance of the haemaglutination reaction; - day 7 of the experiment: drawing blood samples for the procurement of serum; inoculation of 0.5 ml suspension 5% sheep red cells; inoculation of 0.1 ml sheep red blood cell lysate into the wattle; measurement of the inoculated barbs, pre-inoculation (a.i.) and at 24, 48 and 72 h post-inoculation (p.i.); - day 14 of the experiment: drawing blood samples for the determination of some hematological parameters (total red blood cells, hemoglobin, hematocrit) and for the procurement of serum; the performance of the haemaglutination reaction; measurement 353

7 of the inoculated barbs, pre-inoculation (a.i.) and at 24, 48 and 72 h post-inoculation (p.i.); 5 birds from every lot were inoculated with physiological solution as a placebo. The statistic interpretation of the data was carried out through the T test for comparing the means for 2 pair groups, using the soft Statistic 9. The presence or the absence of the correlation (r) between the presence of antibodies against sheep red blood cells was calculated and also the reaction obtained after the IDR was done using Pearson s correlation test with Statistics soft. Results The clinical exam performed at the beginning and during the experiment pointed out a pale comb, wattles and mucous membrane of the hens from the group infested with D. gallinae and normal aspect for the ones from the treated group. The hematological exams performed in the case of the group infested with D.gallinae in the day 0 of the experiment revealed: total red blood cells ±175000/mm 3, hemoglobin - 7,2±0,374 g/dl, haematocrit 24,1±1,69%. In the day 14 of the experiment the following were registered: total red blood cells ±169000/mm 3, hemoglobin - 7,05±0,342 g/dl, haematocrit 23,6±1,49%. In the case of the treated lot in day 0 of the experiment the following were registered: total red blood cells ±127000/mm 3, hemoglobin - 8,51±0,382 g/dl, haematocrit 26,5±1,5%. In the day 14 of the experiment the following were registered: total red blood cells ±165000/mm3, hemoglobin - 8,68±0,34 g/dl, haematocrit 26,4±1,66%. After the monitoring of the egg production a statistically significant difference (p = 0,000002) was noticed between the percentage of the egg laying registered at the hen group infested with D. gallinae (73.2%) and the group that was treated (92.1%). After performing the IDR with sheep red blood cell lysate for the hens inoculated s.c. twice in a week s time with 0.5 ml sheep red blood cell suspension 5% the local reaction was more evident after 48 h p.i. manifested through the appearance of a diffuse edema at the level of the inoculated wattle. The local alterations were lower for the lot infested with D. gallinae. The differences between the wattle thickness of the both lots of hens inoculated with sheep red blood cells were registered at 48 and 72 hours p.i. and 354

8 are statistically significant (p= ), indicating the suppression of the immunity mediated al a cellular level. The wattle edema was not observed at the hens that were inoculated with physiological serum. They only presented a small haematoma localized at the inoculation place. After the performing of the RHA, 7 days after the inoculation of the antigen (0.5 ml sheep red blood cells 5%) we could notice that for the serums that came from the hens from the group that was infested with D. gallinae, the average titre at which the haemaglutination took place was 1/12. The differences between the titres of the antibodies formed against sheep red blood cells sinthetized by the two hen groups were statistically significant (p= ). The average titre at which the HA took place, 7 days after the second inoculation of the antigen was of 1/23 for the hen group infested with D. gallinae and 1/53 for the hens in the treated group. The differences between the titres of the antibodies formed against sheep red blood cells by the two hen groups were statistically significant (p=0.02). The significant differences between the titres of the antibodies formed against sheep red blood cells registered for the two groups during the experiment indicates the suppresion of the immune humoral response in the hens infested with D. gallinae. 3. THE IN VITRO TESTING OF THE SYNTHETIC ACARICIDES EFFICACY ON DERMANYSSUS GALLINAE In this chapter we tested the compared efficacy of three synthetic acaricide molecules (amitraz, deltamethrin and diazinon), derived from three different classes (formamidines, pyrethroids and organophosphrics), on the mite D. gallinae. The samples were collected from bird rearing on the intensive and extensive systems. We also tried to track down and point out the appearance of the chemoresistance phenomenon of the D. gallinae mite, compared to the acaricides used for our study. The research was performed in vitro, during June 2005 September The mites (D. gallinae) were collected from 5 poultry farms with hens kept in battery cages 355

9 and from 15 private households in 5 different areas of the county Cluj (Ocna-Dej, Câţcău, Apahida, Lona and Dăbâca). Like the synthetic acaricides, there were used: amitraz (Taktic), solution 0.25, 0.5, 1 ; deltamethrin (Butox), solution 0.5, 1, 2 ; diazinon (Diazinol), solution 0.5, 1, 2. Straps with the dimension of 10/100 mm were cut from filter paper and soaked in the research substances. Then the strips were dried and introduced in Pasteur pipettes. 3 filter paper straps were deeped for each concentration and there were also 3 filter paper straps impregnated with distilled water, representing the witnesses. For each strip there were 10 µl suspension/cm² used. 20 mites were introduced in each dropper with the help of a vacuum pump. The affiliated part of the pipette was removed in order to avoid the access of the mites in that part, and then the top of the pipette was closed. The number of dead mites was registered at 24, 48 and 72 hours. The analysis of the obtained data was interpreted with the help of an original calculator program (ARP) that determined the lethal concentration 50 (CL 50 ) and the resistance factor (RF). Results In the case of the mites taken from the 5 poultry farms with rearing on the intensive system, the rate of mortality was of 100% after 24 hours of contact and it was signaled at Amitraz used in a concentration of 1 in the farm A (Sibiu county) and E (Cluj county) and for Deltamethrin 2 in the case of the farm D (Cluj county). In the case of the mites taken from private household shelters of the population, after 24 hours of contact, the amitraz 1 determined a 100% rate of mortality in the case of the mites from all 15 shelters; the deltamethrin 2 determined a rate of mortality of 100% in the case of the mites taken from two shelters and the diazon 2 a mortality of 100% in the case of the mites taken from 6 shelters. The CL 50 values in the case of the amitraz were between (0.91) for the mites taken from the bird rearing on the intensive system and between (0.04) the mites from the extensive systems. For the deltamethrin, CL 50 had values between (3.661) for the mites taken from intensive systems and between 356

10 (0.494) for the mites from the extensive systems. In the case of diazinon, CL 50 had values between (1.2) for the mites from intensive bird rearing systems and between (0.196) for the ones from the extensive systems. The CL 50 values were lower in the case of the acaricides testing on the mites taken from the extensive bird rearing systems, which shows that they develop a higher sensitivity towards the acaricides that were studied. The lowest average value of the CL 50 (0.04) was observed for the amitraz in the case of the mites taken from the extensive systems, and the highest average value of the CL 50 (3.661) was observed for the deltamethrin, in the case of the mites from intensive systems. RF in the case of the amitraz, had values between (3.9) for the mites taken from the intensive bird rearing systems, and values between (1.9) for the mites from extensive systems. There was no significant evidence for the chemoresistance phenomenon towards amitraz for the mites populations that were studied. The RF values in the case of the deltamethrin testing were of (33.3) for the mites from the intensive systems, and between (4.4) for the ones from extensive systems. High values of the RF ( ) could be noticed for the mites from for (80%) farms and from for (26.6%) private households ( ). This showed a clear evidence of the mites chemoresistance towards the pyrethroid acaricides. The RF for the diazinon tests had values between (1.2) for the mites from the intensive systems and between (0.196) for the mites from the extensive bird rearing systems. In the case of the mites taken from 3 (60%) farms (RF = ) and from 4 (26.6%) private households (RF = ), the presence of the chemoresistance phenomenon towards the oraganophosphoric products was noticeable. 4. THE IN VITRO TESTING OF SOME PLANT EXTRACTS ON DERMANYSSUS GALLINAE The objective of this chapter was the testing of the in vitro efficacy of 11 vegetal extracts on the ticks D. gallinae, taken from intensive and extensive bird exploiting systems. 357

11 The research was performed during June 2005 and September The farms that were taken into study were from the following: Sibiu, Sălaj and Cluj. The private households were from 5 areas of Cluj county (Ocna-Dej, Câţcău, Apahida, Lona and Dăbâca). The following vegetal extracts have been studied: fir (Abies alba), basil (Ocimum basilicum), thyme (Satureja hortensis), cedar (Cedrus atlantica), coriander (Coriandrum sativum), eucalyptus (Eucalyptus globules), cade (Juniperus comunis), lavander (Lavendula officinalis), lemon (Citrus limon), peppermint (Mentha piperita) and pine (Pinus silvestris). Strips of 10/100mm were cut from filter paper, introduced in Pasteur pipettes, and then soaked into the research extracts. The plant extracts were used in a concentration of 10%, 20% and 30%. 3 filter paper strips were impregnated for each concentration and there were also 3 straps impregnated with distilled water, representing the witnesses. Each strip contained 10 µl suspension/cm². 20 mites were introduced in each pipette with the help of a vacuum pump and then the top of the pipette was closed. The number of dead mites was recorded at 24, 48 and 72 hours after the examination of the droppers with the help of a stereo magnifier (x2.5). The analysis of the obtained data was interpreted with an original computer program (ARP) and the lethal concentration 50 (CL 50 ) was determined. Results The best efficacy on the mites taken from the intensive bird husbandry system was obtained by the coriander vegetal extracts (Coriandrum sativum, CL 50 =7.408), thyme (Satureja hortensis, CL 50 =8.593), basil (CL 50 =10.819) and peppermint (Mentha piperita, CL 50 =13.765). A lower efficacy on the mites taken from intensive bird rearing systems was observed for cade vegetal extracts (Juniperus comunis, CL 50 = ), lemon (Citrus limon, CL 50 = ) and fir (Abies alba, CL 50 =97.24). The best efficacy on the mites taken from the extensive bird exploiting system was obtained by mint vegetal extracts (Mentha piperita, CL 50 =0.063), basil (CL 50 =0.252), coriander (Coriandrum sativum, CL 50 =3.424) and thyme (Satureja hortensis, CL 50 =3.815). A lower efficacy on the mites taken from intensive bird rearing systems 358

12 was observed for lemon (Citrus limon, CL 50 =55.269), eucalyptus (Eucalyptus globules, CL 50 =36.228) and lavender (Lavendula officinalis, CL 50 =27.961). CL 50 had lower values for the testing of vegetal extracts on mites taken from extensive bird rearing systems ( ) compared to the ones registered for the mites from intensive bird rearing systems ( ). 359