SCVMJ, IVX (1)

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1 SCVMJ, IVX (1) SEROLOGICAL SURVEY OF ANAPLASMA MARGINALE (RICKETTSIA) ANTIBODIES IN ANIMALS BY MAJOR SURFACE PROTEIN 5 COMPETITIVE INHIBITION ENZYME-LINKED IMMUNOSORBENT ASSAY Tarek R. Abou-Elnaga., Mona A. Mahmoud., Wafaa A. Osman., Azza S. A Goda Animal Health Department Desert Research Center, Mattarya, Cairo, Egypt. ABSTRACT The prevalence of bovine Anaplasmosis caused by Anaplasma marginale was studied in 360 cattle and buffaloes maintained at small holder's farmers at Matrouh governorate between months of June 2006 to July The incidence of this parasite by using microscopical examination was found to be 74 (37%) and 95 (59.3%) of the examined cattle and buffaloes, respectively. Serologically using rmsp5-celisa, the incidence of antibodies against Anaplasma marginale was in 134 (67%) and 125 (78.1%) of cattle and buffaloes, respectively. The naturally diseased cases subjected to chemotherapeutic treatment using L.A oxytetracycline (200 mg/kg b. wt) showed rapid recovery with an undetectable parasitemia as measured by 28 days post-treatment. In conclusion, Anaplasmosis is a prevalent disease of bovine farms in the examined area. Additionally, the ability of the rmsp5-celisa to accurately detect individually infected animals will facilitate epidemiologic investigations, in areas where the Anaplasmosis expanding through purchasing of infected animals and getting access into disease-free regions. INTRODUCTION Bovine Anaplasmosis is an arthropods-borne hemolytic disease of cattle that is caused by the rickettsia Anaplasma marginale (Kocan, et al., 2003). Anaplasmosis is also called "Yellow bag" or yellow fever as affected animals can develop a jaundiced appearance. It is an important disease as it tends to cause outbreaks in a herd, which can lead to death of adult cattle. Other economics losses include abortions, decreased weight gain, bull infertility and treatment costs (Zaugg and Lincolon, 1995; Luther, et al and Bock, et al., 2003). Anaplasma marginale is the most common pathogen of cattle (Smith, 2002 and Kocan, et al., 2003) and is responsible for substantial economic losses in livestock production of developing countries (Jimenez, et al., 2008). Anaplasma centrale is capable of producing a moderate degree of anemia, but clinical outbreaks in the field are extremely rare. Carrier animals are an efficient source of infection (Eriks, et al., 1993), where they carry Anaplasma species in their

2 310 Tarek et al., bodies, but don't show clinical signs and able to infect other animals Whitter, et al. (2007). Anaplasmosis transmitted by at least 20 tick species besides various other biting arthropods implicated as mechanical vectors. Consequently, the needles used in vaccinations and contaminated surgical instruments make the iatrogenic transmission an efficient way of spreading infection (Brinkman and Kersting, 1990 and Dulmer, et al., 2001). Control of Anaplasmosis requires both an effective vaccine and accurate identification of carrier cattle (Silvia, et al, 2005). A competitive enzyme-linked immunosorbent asssy (celisa) was developed for serological diagnosis of bovine Anaplasmosis with purified recombinant major surface protein 5 (MSP5) (Dreher, et al, 2005). There is few number of studies regarding the epidemiological situation of anaplasmosis in the semi-arid region of northwestern costal belt of Egypt. Despite of the low population of cattle and buffaloes in this area, this activity has an important social impact as subsistence for poor families. This study was therefore designed to determine and compare the prevalence of Anaplasma marginale in tick infested buffaloes and cattle in Matrouh governorate. MATERIALS & METHODS 1-Animals: Three hundreds and sixty cattle and buffaloes of 3 months up to more than 3 years old belonging to farms at Matrouh governorates were randomly selected for blood sampling and clinical examination (Rosenberger, 1979) during the period between June 2006 to July Two hundreds animals were cattle and 160 buffaloes. 2-Samples: Blood and serum samples were collected from all animals for blood smears and serological examination. Serum samples were collected and stored at 20 o c until tested by Competitive Inhibition Enzyme Linked Immunosorbant Assay. 3-Microscopial examination: Blood smears were stained by Giemsa stain and the detection of Anaplasma inclusion bodies were made according to Coles (1986). 4- Serological detection for Anaplasma antibodies (Competitive Inhibition Enzyme Linked Immunosorbant Assay): Animals were tested for antibodies to Anaplasma marginale using the celisa Anaplasmosis kets (VMRD Inc., Pullman, WA, USA) (Knowles, et al., 1996), which detects antibodies to the MSP5 antigen conserved between A. marginale, A. phagocytophilum and A. ovis (Dreher, et al., 2005). Percent inhibition values greater than 30 % were considered positive as recommended by the manufacturer. All samples and controls were run in duplicate and the mean optical density (OD) at 450 nm was determined. The percent inhibition was determined using the mean ODs of each sample compared to the mean of control wells using the formula:

3 SCVMJ, IVX (1) % inhibition = 100 (sample OD X 100) / (negative control OD), as described in the manufacture's protocol. 5- Tick collection and identification: Animals were carefully checked for ticks and their specimens were placed into 70% ethanol in glass vials. Tick numbers were also recorded and identified according to the keys of Manilla (1998). 6- Treatment of infected animals: Twelve animals were used in treatment trial; 6 cows and 6 buffaloes naturally infected with anaplasmosis. Animals were treated with Oxytetracycline / LA at dose rate of 200 mg/kg b.w (I/M injection). Samples were collected frorm all the animals for blood films, serum and packed cell volume (PCV %) pre-treatment day (0), 1 and 4weeks post-treatment. Criteria to assess response to treatment included parasitemia, fever, PCV % and c-elisa as measured before treatment, 7 days and 28 days after treatment and compared with 12 non- infected cattle and buffaloes from the same area. Anaplasma rickettsaemia was calculated as a Percentage of paras-itized erythrocytes (PPE) observed in stained blood films (Bock, et. al., 1997). 7- Statistical analysis: Data were analyzed using the procedure of SAS version 6.12 and Duncan's multiple range tests. (SAS, 1987). RESULTS & DISCUSSION Although tick-borne diseases (TBD) are major threat to livestock, very little work has been done to Anaplasma in buffalo Agigi (1995). In this study, infected animals showed fever, anorexia, severe debility, anemia, and weakness coincided with the same clinical findings reported by Kettle (1995), Imren and Şahal (1996) and De wall (2000). On the other hand, Jiliantai et al. (2009) reported that, no clinical symptoms were recorded in Anaplasma infected cattle. Microscopical examination of blood films (Table, 1) revealed that 37 and 59.3% of cattle and buffaloes were infected respectively, out of which (11.5, 15.5 and 10%) cattle and (21.8.5, 13.1 and 25.6%) buffaloes had Anaplasma marginale, Anaplasma centrale and mixed infection, respectively. This result does not come in accordance with the findings of Khan, et al. (2004), Rajput, et al. (2005) and Ocaido, et al. (2005) as their results were (80%, 36.5% and 30%) in buffaloes and (75.1%, 52% and 63.4%) in cattle, respectively. The results revealed that buffaloes had higher prevailing rate of Anaplasma but the effect is often less serious in comparison to cattle in the same ecosystem Khan, et al. (2004). Serological examination of Anaplasma marginale antibodies (Table, 2) proved that 67% of cattle and 87.1% of buffaloes were positive. A higher and lower rate was recorded by various authors, Cringoli, et al. (2002) (60.9%), De la Fuente, et al. (2005) (50%), Dreher, et al. (2005) (45.1%), Steven et al. (2007) (70.8%) and Junior, et al. (2008)

4 312 Tarek et al., (66.2%) and Kabi, et al. (2008) (58 %). The difference may be attributed to the changes in climatic condition, intensity of tick infestation, and also contaminated needles and instruments transmission is an efficient way of infection spreading(bock, et al. 2003). But the most impor-tant epidemiological factor for the estab-lishment of high prevalence is the persi-stence of infection in the reservoirs (catt-le and buffaloes) (Whitter, et al. 2007). The seasonal distribution is very much related to weather conditions, Table (3) showed that the rise in temperature and humidity had high seroprevalence with a peak in Summer in both cattle (76.5%) and buffaloes (86.1%) due to the abundance and activity of arthropod vector. Similar findings were recorded by Cringoli, et al. (2002), Silvia, et al. (2005) and Dreher, et al. (2005). In Egypt, the native breed of cattle and buffaloes are resistant to ticks. So villagers usually don t worry much about tick infestation. With regards to the survey on the ixodid fauna (Table 4), many potential tick vectors of Anaplasma infection were identified during the investigation in farms with seropositive animals. In this study, three hundred and seventy tick specimens were collected from cattle and buffaloes in Matrouh governorate. Rhipicephalus species was the predominant tick vector (46.2%) fallowed by Boophilus species (29.7%) and finally Hyalomma species (24.1%). Similar findings have been obtained by several authors Keleş, et al. (2001) Cringoli, et al. (2002) and De la Fuente, et al. (2004). Concerning the seroprevalence of Anaplasmosis in different age groups, the results in Table (5) showed that adult animals (more than 3 years) of both cattle and buffaloes had the highest infection rate. This might be explained by the fact that; the age resistance which may lasts up to 12 months and as the animals get older, become more susceptible to infection. Young animals (3-11 months) in cattle and buffaloes showed also high infection rate, this may be due to maternal antibodies in the colostrum (Kvein, et al., 2005). Our results cleared that Anaplasmosis is a disease of adults; a parallel findings were recorded by Hungerford and Smith (1997), Keleş, et al. (2001), Chahan, et al. (2005), Whitter, et al. (2007) and Christine (2008). PCV % provides a simple, quick and accurate mean of detecting one of the most important features of tick-borne diseases anemia (Jain 1993). Rectal temperature is representative of the core body temperature; fever is one of the most important clinical signs of tickborne diseases. In order to study the therapeutic effects of single parenteral injection of long-acting oxytetracycline on cattle and buffaloes infected with Anaplasma maraginale using a separate syringe for each animal, results (Table 6 and7) indicate that the use of long-acting oxytetracycline minimized the severity

5 SCVMJ, IVX (1) of clinical signs of Anaplasmosis as it stoped the increase in infected RBCs before it reach critical level. After treatment the differences of body temperature, PCV % and parasitemia as compared to those before treatment revealed increased value of PCV % and decreased body temperature as well as parasite in Giemsa staining blood films or in c-elisa was not detected. Results given in the present study in terms of treatment are parallel with those of Zaugg and Lincolin (1987), Özlem et al., (1988) and Keleş, et al. (2001) ), Smith (2002), Chahan, et al. (2005), Whitter, et al.(2007) and Christine (2008). CONCLUSION The results highlighted that Anaplasma marginale and their potential vectors are extremely common in the examined area and pointed out the likely risks for animals that are imported from tick born disease (TBD) free areas to upgrade local breeds and suggesting the need to develop a vaccine for the control of infection with potential impact to animal health. Oxy-tetracycline can be fed during the vector season to effectively prevent transmi-ssion to susceptible animal. Table (1): Prevalence of Anaplasmosis in cattle and buffaloes using thin blood film examination. Animal Number of type samples Number of positive samples Total A.marg. A. cent. Mixed No. % Cattle (11.5%) 31 (15.5%) 20 (10%) Buffaloes (21.8%) 21 (13.1%) 39 (25.6%) Total (16.1%) 52 (14.4 %) 59 (16.3%)

6 314 Tarek et al., Table (2): Comparison of Anaplasmosis rate in cattle and buffaloes using celisa. celisa Cattle (No.=200) Serum samples Buffaloes (No.=160) Positive Negative Positive Negative Anaplasma sp. 134 (67%) 66 (33%) 125 (78.1%) 35 (21.9%) Table (3): Seasonal Prevalence of Anaplasmosis among cattle and buffaloes using celisa. Season/animal Cattle Buffaloes Autumn Winter Spring Summer Number Positive % Number Positive % Total

7 SCVMJ, IVX (1) Table (4) Tick Species collected in farms serologically positive to Anaplasma sp. Tick Species (Adult specimens) No. of No. of Total No. (%) male female Rhipicephalus turaicus (35.1%) R. sanguineus (11.1%) Hyalomma anatolicum (16.2%) H. dromaderii (7.8%) Boophilus sp (29.7%) Total Table (5): Prevalence of Anaplasmosis in different age groups of cattle and buffaloes using celisa. Animal type Age groups 3-6months 7 12months 1-3 years More than 3 years All Cattle no.= Anaplasma sp.+ve Buffaloes no.= Anaplasma sp+ve

8 316 Tarek et al., Table (6): Effect of cattle treatment with single dose of L.A oxytetracycline (200 mg/kg b.w ) Parameters Control n = 6 Before Treatment n = 6 After treatment 1 week 4 weeks Mean PCV ±SE 34.46± 2.10 a 22.67± 4.92 d 27.20±1.43 c 33.01± 2.56 b Mean temperature o C ±SE 38.32± 0.22 c ± 0.61 a 39.30± 0.75 b 39.11± 0.27 b Mean parasitemia ±SE - 10± 2.00 a 6.08± 0.64 b - a b c and d Denotes significant differences in comparison to their respective control at p 5 - SE=stander error Table (7): Effect of buffaloes treatment with single dose of L.A oxytetracycline (200 mg/kg b.w). Before Control After treatment Parameters Treatment n = 6 n = 6 1 week 4 weeks Mean PCV ±SE 35..1± 0.34 a 27.67± 1.30 b 28.20±0.46 b ± 0.94 a Mean temperature o C ±SE 38.22± 0.22 b ± 0.91 a 39.00± 0.75 a 38.51± 0.99 b Mean parasitemia ±SE - 11± 0.07 a 7.15± 0.64 b - a b c and d Denotes significant differences in comparison to their respective control at p 5 - SE=stander error

9 SCVMJ, IVX (1) REFERENCES Agigi, M. (1995): The prevalence of blood parasites in cattle in vicinity of Samsun. Journal of Etlik Veterinary Microbiology, 8, 1, Bock, R.E.; D Vos, A.J.; Kingston, T.G. and Mclellan, D.J. (1997): Effect of breed of cattle on innate resistance to infection with Babesia bovis, B. Bigemina and Anaplasma marginale. Am. Vet. J. 75(5) Bock, R.E.; D Vos, A.J., Kingston, T.G. and Carter, P. D (2003): Assessment of a low virulence Austuralian isolate of Anaplasma marginale for pathogenicity and transmissability by Boophilus microplus. Vet. Parasitol. 118: Brinkman, M. H. and Kersting, K. W. (1990): Bovine anaplasmosis: an overview. Iowa State University Veterinarian 52: Chahan, B., Zijian, J., Xuenan, X., Yukita, S., and Hisashi, I. (2005): Serological evidence of infection of Anaplasma and Ehrlichia in domestic animals in Xinjiang Uygur Autonomous Region area, China. Vet. Parasitol. 134, Coles, E. H. (1986): Veterinary Clinical Pathology. 4 th Ed. W.B. Saunders Co. Cringoli, G., Otranto, D., Testini, G., Buono, V., and Puccini, V. (2002): Epidemiology of bovine tick-borne diseases in southern Italy. Vet. Res.33: Cristine, N. (2008): Preventing bovine Anaplasmosis. Personal Communication. De la Fuente, J., Vicente, J., Höfle, U. (2004): Anaplasma infection in freeranging Iberian red deer in Spain. Vet. Microbiol.100: De la Fuente, J., Alessanadra T., Santo, C., and Katherine, M. (2005): Serologic and molecular characterization of Anaplasma species infection in farm animals and ticks from Sicily. Vet. Parasitol. 133: De Wall, D. T. (2000): Anaplasmosis control and diagnosis in South Africa. Ann N. Y. Acad. Sci. 916: Dreher, J. S., De la Fuente, J., Hofman-Lehmann, R., Meli, M. L., and Lutz, H. (2005): Serologic cross-reactivity between Anaplasma marginale and Anaplasma phagocytophilum. Clin. Diagn. Lab. Immunol. 12 (10): Dumler, J. S., Barbet, A. F. Bekker, C. P. J. and F. R. Rurangirwa. (2001): Reorganization of the genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia

10 318 Tarek et al., with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and "HGE agent" as subjective synonyms of Ehrlichia phagocytophila. Int. J. Syst. Evol. Microbiol. 51: Eriks, I.S., Stiller, D., and Palmer, G. H. (1993). Impact of persistent Anaplasma marginale rickettsemia on tick infection and transmission. J. Clinical Microbiology. 31: Hungerford, L.L. and Smith, R.D. (1997): Variations in seroprevalence and host factors for bovine anaplasmosis in Illinois. Vet. Res. Commun. 21(1) İmern, HY., and Şahal, M. (1996): Veterinary Internal Diseases 4 th edition, Medisan press, Ankara. Jiliantai, Nobutaka Seino, Daisuke H., Hiroshi and Hisashi Inokuma (2009): Molecular Survey for Anaplasma bovis and Anaplasma phagocytophilum Infection in Cattle in a Pastureland Where Sika Deer Appear in Hokkaido, Japan. Jpn. J. Infect. Dis., 62, 73-75, Jimenez, R. O., Sergio, D., Rodrguez, C., and De la Fuente, J (2008): Anaplasma marginale: analysis of variable fragment sequences in msp 1 and msp 4 genes in four new Mexican strains. Tec Pecu Mex, 46 (1): Junior, D. G., Araujo, F. R., Silva, F. S. N., Rangel,C. P., and Fonseca, A. H. (2008): Frequency antibodies to Babesia bigemina, B. bovis, Anplasma marginale, Trypansoma vivax and Borrelia burdgorferi in cattle from the Northeastern region of the State of Para, Brazil. Brazil. J. Vet. Parasitol. 17, 2, (2008). Kabi, F., Magona, J. W., Nasinyama, G. W. and Walubengo, J. (2008): Tickborne infections among Nkedi Zebu and Ankole cattle in Soroti district, Uganda. J. Protoozool. Res. 18, Keleş, Ihsan, Serdar, D. Nuri, A. Mehmet, K and Cihangir, A. (2001): Tick-borne diseases in cattle: Clinical and haematological findings, diagnosis, treatment, seasonal distribution, breed, sex and age factor and the transmitters of the diseases. YYU. Vet. Fak. Derg.12 (1-2): Kettle, DS. (1995): Medical and Veterinary Entomology, 2 nd Addition, CAB International, UK. Khan, M. Q., A., Zahoor, M. and Jahangir, Ashraf Mirza, M. (2004): Prevalence of blood parasites in cattle and buffaloes Pakistan Vet. J., 24(4): Knowles, D. P., Torioni D. Echaideand McElwain, T.F. (1996): Antibody against an Anaplasma marginale MSP5 epitop common to tick and erythrocytes stages identifies persistently infected cattle. J. Clin.Microbiol. 34, Kocan, K. M., De la Fuente, J and L. G. Stratton. (2003): Co-feeding studies

11 SCVMJ, IVX (1) of tick infected with Anaplasma marginale. Vet.Parasitol. 112: Kvein, K. L., Junzo, N., Mitchell, S. A., Guy, H. P. and Wendy, C. B. (2005): The CD4+ T cell immunodominants Anaplasma marginale major surface protein 2 stimulate γδ T cell clones that express unique T cell receptors. J. of Leukocyte Biology Vol. 77, February Luther, GD, Hart LT, and Todd WJ. (1996): Anaplasmosis treatment and control. TNVAC Proceeding. Manilla, G., (1998): Acari Ixodida, Edizioni Calderini, Bologna, pp Ocaido, M., Otim, C. P., Okuna, N. and Monrad, J., (2005): Socio-economic and livestock disease survey of agropastoral communities in Serere County, Soroti District Uganda. Livestock Research for Rural Development 17 (8). Özlem, MB., Karaer, Z., Turgut, K., and Ϊnci, A. (1988): Efficacy of long acting oxytetracycline on ovine anaplasmosis. J. Fac. Vet. Med. Ankara Uni. 35: 1, 1-5. Rajput, Z. I., Song-hua, HU., Arijo, A. G., Habib, M., Khalid, M. (2005): Comparative study of Anaplasma parasites in tick carrying buffaloes and cattle. Journal of Zhejiang University Science 6B (11): Rosenberger, G. (1979): Clinical examination of cattle. Verlag Paul Parey. Berlin and Hamburg. SAS (1987): Statistical Analysis, Version 6.03 SAS User's Guide: Statistics SAS Institute Inc. Cary North Carolina U.S.A. Silvia, L. B., Claudio, R. M., and Raul, H. K. (2005): Serlogigcal survey of Babesia bovis, B. bigemina and Anaplasma marginale antibodies in cattle from the semi-arid region of the state of Bahia, Brazil by Enzyme-linked immunosorbant assays. Mem. Inst. Oswaldo Cruz. Rio de Janeiro, Vol.100(6): Smith, B.P. (2002): Anaplasmosis. 3 Animal Internal Medicine Steven, B., Spickett, F., Vosoloo, W. and Duplessis, G. (2007): Influence of dipping practices on the Seroprevalence of Babesiosis and anaplasmosis in the Foot and mouth disease buffer zone adjoining the Kruger National Park in South Africa. Onderstepoort Journal of Veterinary Research 74: Whitter, D., Nancy, C. and John, F. C. (2007): Anaplasmosis in Beef cattle. Virginia State University Publication No , posted July Zaugg, JL., Lincoln, SD. (1995): Protection enhancement against bovine anaplasmosis by simultaneous administration of Propionibacterium acnes and a commercial vaccine. Agri-Practice, Nov./ Dec V. 16, No. 10.

12 320 Tarek et al., الملخص العربى مسح سيرولوجى لألجسام المضادة ألنابالزما مارجينال )ريكتسيا( فى الحيوانات باستخدام األليزا التنافسية. طارق رمضان أبو النجا منى عبد هللا محمود وفاء عبد اللطيف عثمان- عزة سعيد أحمد جودة قسم صحة الحيوان مركز بحوث الصحراء- القاهرة. عند دراسة مدى انتشار األنابالزما مارجينال بين األبقار و الجاموس فى محافظة مطروح فى الفترة من شهر يونيو 6002 الى يوليو 6002 تبين أن % 72 من األبقار و % 3.97 من الجاموس التى تم فحصها ميكروسكوبيا مصابة بالمرض. بينما تبين من التشخيص السيرولوجي باستخدام األليزا التنافسية أن نسبة األصابة % 22 و % 2.97 بين األبقار و الجاموس على التوالى. استخالص النتائج التالية من الدراسة:- تم عالج الحاالت المصابة بنجاح باستخدام عقار األوكسىتتراسيكلين. انتشار األنابالزموسيس بمنطقة البحث, وقد تم قدرة اختبار األليزا التنافسية على تشخيص األنابالزما مارجينال بدقة بشكل منفرد مما يسهل دراسة وبائية المرض فى المناطق التى تنتشر فيها األنابالزما مارجينال عن طريق شراء حيوانات مصابة يتم ادخالها لمناطق خالية من المرض.

SEROPREVALENCE OF ANAPLASMA OVIS ANTIBODIES IN SMALL RUMINANTS BY MAJOR SURFACE PROTEIN 5 COMPETITIVE INHIBITION ENZYME-LINKED IMMUNOSORBENT ASSAY

SCVMJ, IVX (1) 2009 287 SEROPREVALENCE OF ANAPLASMA OVIS ANTIBODIES IN SMALL RUMINANTS BY MAJOR SURFACE PROTEIN 5 COMPETITIVE INHIBITION ENZYME-LINKED IMMUNOSORBENT ASSAY Azza S.A Goda., Wafaa A. Osman.,