Experimental Study about Canine parvovirus ...

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‫والبيطرية‬ ‫النشر‬ ‫االطروحة للعلوم الزراعية شروط‬

‫العدد الخامس ‪0281 /‬‬

‫تنشر اجمللة البحوث العلمية يف اجملاالت املعرفية املتعلقة جبميع العلوم يف طبعاتها‬‫املتخصصة مبنهجية البحث العلمي وخطواته املتعارف عليها عامليا باللغتني العربية‬ ‫واالنكليزية على ان تكون مطبوعة بنظام (‪ )word2007‬ومل يسبق نشرها‪ ،‬ويف حالة قبوله‬ ‫جيب اال ينشر يف اية دورية من دون اذن كتابي من رئيس حترير الطبعة املتخصصة ‪.‬‬ ‫ جيب اال يزيد البحث يف مجيع االحوال عن (‪ )61‬عشرين صفحة بضمنها املراجع واحلواشي‬‫واجلداول واالشكال واملالحق‪ ،‬ويكون نوع احلرف املستخدم (ايريال–‪ )Arial‬حجم احلرف‬ ‫للبحث (‪ )01‬وللعنوان الرئيس ‪ 61‬بولد والسم الباحث ‪ 02‬بولد والسم الكلية واجلامعة ‪01‬‬ ‫بولد‪ ،‬وتكون اهلوامش واملصادر يف نهاية البحث ألسباب تتعلق بالتصميم حبجم ‪ 06‬بولد‪ ،‬ويف‬ ‫حال زيادة البحث عن عدد الصفحات املقررة يتحمل الباحث ‪ /‬الباحثة فرقا يف االجور عن‬ ‫كل صفحة زيادة مببلغ ‪ 6011‬دينار‪ ،‬اما الصفحة امللونة فتكون اجورها مخسة آالف دينار‬ ‫للعراقيني‪ ،‬وللعرب واالجانب مخسة دوالرات‪.‬‬ ‫ تنشر البحوث االنسانية باللغة العربية مع ملخص باإلنكليزية‪ ،‬اواإلنكليزية مع ملخص‬‫بالعربية ‪.‬‬ ‫ تنشر البحوث العلمية باللغة االنكليزية فقط مع ترمجة للعنوان الرئيس واسم الباحث‬‫ولقبه العلمي واسم الكلية واجلامعة بالعربية ‪.‬‬ ‫ يكتب عنوان البحث باإلنكليزية ايضا ان كان البحث بالعربية ‪.‬‬‫ تكون اخلالصات مطبوعة باللغة االنكليزية فضال عن وجودها باللغة العربية يف البحوث‬‫االنسانية‪ ،‬و باللغة العربية فضال عن وجودها باإلنكليزية بالنسبة للبحوث العلمية ‪.‬‬ ‫يتعهد الباحث (خطيا) بان حبثه من نتاجه العلمي والفكري وهو خاضع ملعايري األمانة‬‫العلمية وضوابط االقتباس ولَم يستله كليا او جزئيا من حبث او رسالة او أطروحة علمية‬ ‫او أي نتاج فكري او علمي لباحث اخر ولَم يسبق نشره يف جملة اخرى وفِي حال ثبوت العكس‬ ‫يتحمل مجيع التبعات القانونية والفكرية كافة وحيظر التعامل معه ويتم ابالغ املؤسسة‬ ‫او اجلامعة اليت يعمل فيها رمسيا ويذكر يف التعهد الذي يقدمه للمجلة امسه الكامل‬ ‫ولقبه العلمي واسم الكلية اواملعهد واجلامعة اواملؤسسة اليت يعمل فيها مع رقم هاتفه‬ ‫النقال‪.‬‬ ‫ ترسل البحوث على الربيد االلكرتوني للمجلة‪:‬‬‫‪[email protected]‬‬ ‫‪[email protected]‬‬

‫‪5‬‬

‫االطروحة للعلوم الزراعية والبيطرية‬


‫اجور النشر ‪:‬‬‫‪ - 0‬حبث االستاذ ‪:‬‬ ‫أ ‪ -‬للعراقيني ‪ 011 :‬الف دينار‬ ‫ب ‪ -‬للعرب واالجانب‪ 001 :‬دوالرا‬ ‫‪ - 6‬حبث الرتقية العلمية‪:‬‬ ‫للعراقيني‪ 060 :‬الف دينار‪.‬‬ ‫للعرب واالجانب‪ 611 :‬دوالر او ما يعادهلا باليورو‬ ‫‪ - 2‬اجور نشر (حبث مستل) لطالب الدراسات العليا لغاية ‪ 00‬صفحة‪.‬‬ ‫أ ‪-‬للعراقيني ‪ 50 :‬الف دينار‬ ‫ب ‪ -‬للعرب واالجانب ‪ 50 :‬دوالرا‬

‫ تسديد االجور ‪:‬‬‫‪ ‬يكون التسديد ملن هو خارج العراق حبوالة بنظام ( ‪ )Western Union‬باسم‬ ‫املشرف العام ‪.)IBRAHIM ZAIDAN KHALAF( :‬‬ ‫‪ ‬يكون التسديد حبوالة ملن هو يف بغداد اوخارجها باسم املشرف العام (ابراهيم زيدان‬ ‫خلف)‪ ،‬اونقدا من خالل مراجعة مقر اجمللة يف بغداد ‪ /‬بغداد اجلديدة ‪ /‬جممع‬ ‫سر من رأى (الطابق االرضي) ‪ /‬جماور شركة الطيف للتحويل املالي‪/‬مقابل‬ ‫مطعم النعمان ‪.‬‬

‫مالحظة ‪ :‬يتحمل الباحث رسوم احلوالة ‪.‬‬

‫‪6‬‬



‫مالحظات مهمة جداً‬ ‫ ختضع البحوث لالستالل‬‫حيال البحث اىل حمكمني اثنني ويف حال االختالف يف الرأي حيال اىل استاذ حمكم‬‫ثالث إلبداء الرأي بشأن صالحيته للنشر‪ ،‬ويعاد البحث اىل الباحث للعمل‬ ‫مبالحظات االستاذ احملكم ليكون صاحلا للنشر ‪.‬‬ ‫‪ -‬تعاد االجور للباحث يف حال رفض االساتذة احملكمني للبحث ‪.‬‬

‫للتواصل معنا على هواتفنا ايضا ( فايرب وواتس اب ايضا )‬ ‫‪110215502020100‬‬ ‫‪110215016501600‬‬ ‫او زيارة مقرنا يف بغداد اجلديدة ‪ /‬جممع سر من رأى ( الطابق االرضي) ‪/‬‬ ‫جماور شركة الطيف للتحويل املالي ‪ /‬مقابل مطعم النعمان ‪.‬‬

‫‪7‬‬


‫‪8‬‬


NPK


‫‪11‬‬




‫الصعوبة تكمن يف املدى اذلي تستطيع جملة مثل ( االطروحة) ان حتققه من جناح ويه‬

‫تدخل املجالني العلميني هذين ‪ .‬ولكن االرصار ىلع اصدار الطبعة واثلقة بقدرات من‬ ‫معه اكنا كفيلني بإصدار هذه الطبعة اليت بني ايديكم واليت يراهن اجلميع ىلع جناحها‬

‫ومن اهلل اتلوفيق ‪.‬‬

‫فتحية لرئيس حتريرها (االستاذ املساعد ادلكتور نهاد عبد اللطيف عيل انلداوي) وهو‬

‫يرى ثمرة جهوده وجهود زمالئه اذلين آزروه يف ظهور هذه الطبعة اىل انلور ‪ ،‬ومثلها اىل‬ ‫مجيع اعضاء هيئة حتريرها واستشاريتها من بذل جهدا ومن وعد ووعد احلر دين ‪.‬‬

‫تمنيايت للجميع بانلجاح واتلوفيق وشكرا ملن ساند االطروحة يف مشوارها العليم هذا ‪.‬‬

‫‪12‬‬

0281 / ‫العدد الخامس‬


Effect of Salicylic acid and Water Quality on Yield and NPKcontentsin Spinach ‫تأثري املعاملة حبامض السالسيلك ونوعية مياه الري‬ ‫ لنبات السبانخ‬NPK ‫على حاصل وحمتوى األوراق من‬ ‫ دريد كامل عباس الطائي‬.‫د‬.‫م‬.‫ا‬ ‫ قسم البستنة وهندسة الحدائق‬/ ‫جامعة القاسم الخضراء –كلية الزراعة‬ Assistant Prof. Duraid Kamel Abass Altaey Al-Qasim green uni. , Agriculture College / Horticulture dept [email protected]

Abstract Anexperiment was conducted on a private farm in Babylon Governorate at the period from 1/10/ 2014 to 15/5/ 2015the salinity of irrigation water and salicylic acid treatment with three concentrations (0, 50 &100 mg /liter ) , the irrigation water levels was ( 1.3 , 5 & 10) dS.m-1,the water salinity increased electrical conductivity of the soil , sodium contents in leaves, resulting in decreased growth and leaf contents of NPK, salicylic acidapplications alleviated negative effects of saline water by increasing dry weights of shoots and NPK contents in, reduction of sodium contents in leaves. 13



Key words: Spinach, Oxidative stress, Salinity, salicylic acid, NPK

Introduction High-salinity conditions in agricultural soil and irrigation water is one of the most serious challenges faced by agricultural crops in the world. It is estimated that saltaffected soils impact nearly 10% of the land surface and 50% of irrigated land in the world (Ruan et al., 2010), Secondary salinization from irrigation water is a growing worldwideproblem as more than 6% of agricultural land has become saline, Soil salinity reduces water availability of plant roots via negative (low) osmosis potential, as well as decrease of germination dynamics of plant seeds by ionic toxicity of Na and Cl, Significant differences in fruit-set, yield, photo synthetic rates, stomatal conductance, total chlorophyll content, proline, In general, salinity affects almost every aspect of the physiology and biochemistry of plants (Al-Taey,et al, 2017), the mechanism of plant tolerance in the non-salty plants to be happened by more then way, by avoid absorbed salty ions such as Na or it's have other mechanism involved to discarding the Na and Cl from cells and keep on high levels of K in the cells, the other mechanism involved with the cell ability of osmoregulation by raised the osmotic pressure of cell to obverse the outer osmotic pressure and absorb the water from the soil solution (Alzubaidi, 1989). Salicylic acid (SA) and related compounds have been reported to induce significant effects on various biological 14



aspects in plants. These compounds could be influence in a variable manner; inhibiting certain processes and enhancing others (Al-Taey, 2009, ALTaey and Saadoon 2014). (Khodary 2004)proved that, salicylic acid enhanced the maize salt tolerance in terms of improving the measured plant growth criteria. Salicylic acid appears to stimulate maize salt tolerance by activating the synthetic process, some researches mentioned to have relation between the Salicylic acid and the tolerance of salt stress in plants. There are more suggestions to explain mechanism of Salicylic acid for increasing the plant tolerance to salt stress; however the way of signal regulation of plant resistance to unfavorable factors of environment induced by SA are still not clear (Shakhabutdiovaet.al. 2003).

Material and Methods The research was conducted in a field that located at AlMuradia \ Babylon Governorate during the winter growing season of 2012-2013, the broad been seed was a local Iraqi cultivar, many soil samples taken from the field randomly to analysis some physical and chemical characters as show at table (1), the chemical drainage water characters as show table (2),After the seed soaking in salicylic acid , inside plots (2 * 3 m) as lines the distance between them 30 cm and between the plants 10 cm the total was 200 plants to each plot. The experiment included studying the effect of water quality with three qualities, seeds soaking of Salicylic acid with three levels and interaction between them to improve the growth, 15



yield and some chemical characters by testing the role of Salicylic acid to reduce the negative effects by using the Saline drainage water included three levels (1.3, 5 & 10 dS.m-1), The drainage water was mixing by river water to got 5 & 10 dS.m-1 by 2 basins in the ground; the capacity to each one was 10000 liter, The treatments; the salicylic acid application (0,50, 100 mg/L),The experiment conducted as factorial experiment by split plot design with 9 treatments, each one had three replicate comparison was done using Least significant difference ( L.S.D) to compare means with probability of 0.05 level. Table (1) some physical and chemical characters of study soil. Soil separates

Texture

0.42

Sand Silt

%

Loam

0.33 0.25

Clay Chemical characters

unit

Ec

dS-1

2.8

pH

‫ــــ‬

7.22

meq /L

69.21

Soluble ions Ca++

16



Mg++

meq /L

8.73

Na+

meq /L

7.44

K+

meq /L

0.52

Co3=

meq /L

HCO3-

meq /L

0.71

Cl-

meq /L

11.87

SO4=

meq /L

13.56

Table (2) the chemical analysis of drainage water. The drainage water analysis Ca++

Ec pH dS-1

Mg++

Na+

K+

Co3=

HCO3-

Cl-

SO4=

mmole /L

mmole/L

mmole/L

mmole/L

mmole/L

mmole/L

mmole/L

mmole/L

5

6.76

27.74

17.11

38.75

0.48

0.81

27.21

26.41

10

6.8

47.15

38.18

44.09

0. 54

0.65

59.91

62.61

Study characters  The dry weight roots.  The total of vegetative yield (ton/ hectare), the total yield was calculated by made two cuts, the first cut at 50 day after sowing and the second cut at 75 day after sowing.

17



 The leaves contents of Nitrogen: the determination of N by Microkjeldahl (Jackson, 1958).  The leaves contents of Sodium & potassium : It was determination by Flame photometer according to (Weissman&Nehring, 1960).  The leave contents of phosphor: Phosphorus was determined using the Ammonium molybdate by spectrophotometer at wavelength 882 nm (Page, et al., 1982).

Result and Discussion The yield and dry weight of shoot : The results presented at tables (3,4) showed a significant differences between the means ,the treatment which irrigated with 1.2 dS.m-1 recorded the heights averages of yields , while the treatments which irrigated 10 dS.m-1 recorded lower means of yields the dropping ratio was (27% and 48%) in salinity levels 5 and 10 dS.m-1, respectively. The inhibitory effects of salinity on growth of Spinach are probably due to decreased water absorption and disturbed metabolic processes leading decreased meristematic activity or cell enlargement (Kaydan and Okut, 2007; AlTaey, et al., 2015), there are two ways which salinity retards growth, by damaging growth cells so that they cannot perform their functions or by limiting their supply of essential metabolites (Al-Taey, et al, 2017).There is no significant value with both of salicylic acid concentrations (50, 100 mg /L) compared with control as for total yieldwhile its achieved significant value as for dry weight of shootsthis increasing may due to 18



the ability of salicylic acid to improve the plant tolerance by promotes the formation of ROS in the photosynthetic tissues of plants during salt stress and osmotic stress (AlTaey&Saadoon, 2014) , or may the reason of that Salicylic acid provided protection against salinity in plants, probably due to the increased activation of aldose reductase and APx enzymes and to the accumulation of osmolytes, such as sugars, sugar (Al-Azawi,2015) Same data at table (4) presented a significant differences between the interaction between the water quality &Salicylic acid , the best treatments was irrigated with 1.2 dS.m-1 and treated with Salicylic acid whether 50 or 100 mg.L-1 while the lower means was treatment which irrigated with 10 dS.m-1 untreated with Salicylic acid. Table (3) Effect of water quality, Salicylicacid treatment on treatment on yield (ton .hr-1) Treatments Salicylic acid conc.mg.L-1

Effect of water quality

Water Quality 0

50

100

1.2 dS.m-1

11.323

11.654

11.543

11.506

5 dS.m-1

8.432

8.742

8.323

8.449

10 dS.m-1

5.564

5.453

5.654

5.545

Overlap effect Sig 0.05 = 0.446 Salicylic acid effect

8.433

8.612

Sig 0.05 = N.S

19

Sig 0.05 0.405

8.506



Table (4) Effect of water quality, salicylic acid treatment on shoot dry weight (g). Treatments Salicylic acid conc.mg.L-1 Water Quality


0

50

100

1.2 dS.m-1

8.765

9.543

9.432

9.246

5 dS.m-1

7.098

7.546

7.664

7.436

10 dS.m-1

5.432

5.401

5.654

5.495

Salicylic acid effect

Overlap effect Sig 0.05

Sig 0.05

= 0.551

0.435

7.099

7.496

7.583

Sig 0.05 = 0.292

NPK contents in leaves Results in the tables (5, 6,and 7) showed that reduction in NPK contents in leaves with an elevation of water salinity especially in 10 dS.m-1significantly , the reduction may be by reason of the high ions concentration in the soil solution, that affected on the activation of the enzyme inside the plant cells via inhibited the active sites of enzymes and due to metabolism disorder by H+-ATPase pumps break down or via inhibited the membranes working, that affected on the permeability of its as well as the salinity affected on the Photosynthesis, Respiration processes and Electron transport chains (Orcutt, 2000 ), Conversely the Salicylic acid application increased the NPK contents in leaves but 21



the elevations was insignificantly in K contents in leaves, The reason may be due toInduction the enzymatic antioxidants system by increased the SOD, CAT, POX activity and reduced the oxidation of photosynthesis pigments (Yang, et al.,2004), or may be The application of exogenous salicylic acid appeared to induce a pre-adaptive response to salt stress, leading to the promotion of protective reactions to the photosynthetic pigments and the maintenance of membrane integrity in barley plants, which was reflected in an improvement in plant growth (El Tayeb,2005; Al-Taey et al., 2010) Table (5) Effect of water quality, salicylic acid treatment on N% in leaves. Treatments Salicylic acid conc.mg.L-1


Water Quality 0

50

100

1.2 dS.m-1

3.654

3.686

3.576

3.638

5 dS.m-1

3.143

3.053

3.271

3.155

10 dS.m-1

2.680

2.713

2.810

2.734

Overlap effect Sig 0.05 0.231 Salicylic acid effect

3.159

3.150

Sig 0.05 = 0.054

21

Sig 0.05 0.123

3.219



Table (6) Effect of water quality, salicylic acid treatment on K% in leaves. Treatments


Salicylic acid conc.mg.L-1 Water Quality 0 1.2 dS.m-1

2.554

50

100

2.765

2.730

2.683

5 dS.m-1

1.729

1.897

1.792

1.806

10 dS.m-1

1.637

1.553

1.562

1.584



Sig 0.05

=0.434

0.297

1.973

2.071

2.028

Sig 0.05 = N.S

Table (7) Effect of water quality, salicylic acid treatment on P% in leaves. Treatments


Salicylic acid conc.mg.L-1 Water Quality 0

50

100

1.2 dS.m-1

1.563

2.187

2.207

1.986

5 dS.m-1

1.047

1.207

1.237

1.163

10 dS.m-1

0.550

0.853

0.900

0.768



Sig 0.05

= N.S

0.5569

1.053

1.416

Sig 0.05 = 0.157

22

1.448



Reference 1. Al-Azawi,

S.S.M.2015. Effect of Water Quality and Salicylic acid Treatment on Growth and Catalase Activity of Maize Seedlings (Zea mays L.) Journal of Babylon University/Pure and Applied Sciences; (4) (23) p: 1667-1685. 2. AL-Taey, D.K.A and A.H. Saadoon. 2014. Effect of treatment of Salicylic acid and water Salinity on the Growth, and Nitrate Accumulation with nitrate reductase activity in the Leaves of Spinach, Spenaciaoleracea L.Journal of Babylon University/Pure and Applied Sciences;3 (22) p: 1188-1203 3. Al-Taey, D.K.A., 2009 .Effect of spraying acetyl salicylic acid to reduce the damaging effects of salt-water stress on orange plants (Citrus sinensis L.). Journal of Kerbala University; 7 (2) P: 192-202. 4. AL-Taey, D.K.A., A.S. H. Al-Janabi and A.M. Rachid .2017. Effect of water salinity, Organic and minerals fertilization on growth and some nutrients elements in cabbage Brassica oleraceavarapitate. Babylon journal of Pure and Applied science, 25(6):232- 248. 5. Al-Taey, D.K.A., S.S.M. AlAzawi and M.H..Husien. 2009.Effect of Spraying Acetyl Salicylic Acid on the Plant Tolerance for Salt Stress & Survival Percentage after Transplanting of Orange (Citrus sinensis).Babylon journal university -Pure and Applied science;18 (4) ;:1513-1520 . 6. Alzubaidi A.H., (1989). Soil salinity, the applications and thyroidal bases. Ministry of higher education .Iraq.1989 7. El Tayeb, M. A., (2005). Response of barley grains to the interactive effect of salinity and salicylic acid. Plant Growth Regul, 45: 215-224. 8. KaydanD, and M.Y. Okut .2007. Effects of salicylic acid on the growth and some physiological characters in salt stressed wheat (Triticumaestivum L.). TarimBİlimleriDergisi, 13(2): 114-119. 9. Khodary, S.E.A. (2004). Effect of salicylic acid on the growth, photosynthesis and carbohydrate metabolism in salt stressed Maize plants. International Journal of Agriculture& Biology. Vol. 6, No. 1, 2004. 10. Orcutt, D.M., & E.T. Nilsen (2000). The physiology of plants under stress. USA. 11. Ruan CJ, da Silva JAT, Mopper S, Qin P, Lutts S. Halophyte improvement for a salinized world. Critical Reviews in Plant Sciences. 2010;29:329–359. 12. Sakhabutdinova, A.R., Fatkhutdinova, D. R., Bezrukova. M. V. & F. M. Shakirova. (2003). Salicylic acid prevent the damaging action

23



of stress factors on wheat plants. Institute of Biochemistry and Genetics, Ufa Scientific Centre Russian Academy of Sciences. BULG. J. PLANT PHYSIOL., SPECIAL ISSUE, 314 –319. 13. Wiessmann, H. and K. Nehring .1960. Agriculture Chemical analysis Gsm methods for fertility and animal feed, boden und milek. drittevoellig. neubeasrbeiteteauflageverlagpaulparey. Hamburg und Berlin 14. Wiessmann, H. and K. Nehring .1960. Agriculture Chemical analysis Gsm methods for fertility and animal feed, boden und milek. drittevoellig. neubeasrbeiteteauflageverlagpaulparey. Hamburg und Berlin 15. Yang, Y., Qi, M., and Mei, C. 2004. Endogenous salicylic acid protect rice plants from oxidative damage caused by ageing as well as biotic and abiotic stress. Plant J., 40: 909-919. 16. AL- Taey, D.K.A., A.S. H. Al-Janabi and A.M. Rachid, 2015. Effect of irrigation water quality and organic and mineral fertilization on the availability of some nutrient elements and cabbage yield (Brassica oleracea var. capitate L.), Euphrates Journal of Agriculture Science; 7(4) p: 235-247. 17. Page, A.L., R. H. Miller and D. R. Keeney .1982. Methods of soil and analysis Part 2, 2nd ed, Agron. 9, Madison, W. 18. Jackson, M.L. 1958. Soil chemical analysis. Prentice Hall. Englewood Cliffs. N.J.

24

Experimental Study about Canine parvovirus isolated in Iraq

... / / / Hussein A. Mohammed Al.Bayati Microbiology Dept. / Vet. Med College / Wasit University .Iraq BVMS. MSc. PhD (Virology)

Abstract Canine parvovirus2 was known for causing a more severe symptoms in pups. The

experimental

challenge

was

successfully performed by using highly infectious fecal samples that which used for isolation subsequently , then inoculated in two groups of local breed dogs.The clinical signs were observed daily and virus shedding detected by rapid antigen test kit besides checking of total and differential white blood cells count, the clinical signs which included mucoid diarrhea, rough coat, sunken eyes and the virus shedding in feces started at 72 hours and there were leukocyte count variations of two groups. Keywords: Canine parvovirus , experimental , Iraq

Introduction "Canine parvovirus (CPV)2 is responsible for a severe, highly contagious gastroenteric disease in pups. The canine parvovirus 2 (CPV-2) belongs to the family Parvoviridae. The genome consists of a single molecule of linear single stranded DNA, 5.2 Kb in size. It is a non-enveloped, icosahedral symmetry virus whose single stranded DNA encodes two capsid proteins (VP1 and VP2) and two nonstructural proteins (NS1 and NS2). VP1 and VP2 are translated from alternative splicing of the same RNA [1] VP2 mainly comprises the icosahedral capsid of CPV, and only a few amino acid substitutions in its sequence can alter relevant biological characteristics to the virus [2]". Leukopenia may be present with death occurring as little as two days after onset of disease.

There is sepsis and

electrolyte disturbances, hypoglycemia and central nervous system (CNS) hemorrhage from disseminated intravascular coagulation (DIC) may cause neurological disease [3,4,5] . Myocarditis may develop due to inutero exposure or under eight weeks of age[3,6]. Asymptomatic urinary tract infection developed in 25% of puppies following parvovirus enteritis [7]. The disease can occur in dogs at any age or sex or breed, the disease can

transmits by oronasal exposure to contaminated feces, hair coat and fomits including instruments, insects, and rodents are responsible for spreading of the disease. The virus can survive in the environment for months or years. [7]

Materials &Methods Experimental design and infection The current study was done by using of two groups of local breed dogs, each group contain five dogs: first group which include dogs at age range from 1-3 months. While

the

second group include dogs at the same age 25 days. In these two groups using two methods of inoculation as follows: First group was infected by fasting 24 hours and administered of fecal suspension of highly infectious sample which is used in viral isolation respectively , after that was gave food (oral route of infection), and show every day the progression

of clinical signs, then collected of blood

samples to detect of leucopenia, checking of virus shedding using rapid monoclonal antibody test [8]. Second group not fasted but use I/M injection of 0.5 ml leptospiral

bacterin

and

then

administered

of

fecal

suspension and followed by finding same points above , this

which leads to increased severity of parvoviral disease due to immune suppression [9].

Results & Disucusion Ten pups were obtained from field of

unvaccinated and

unexposed bitchs . All pups were showed healthy at the begining of experiment. First group contain five dogs were challenged orally with highly infectious samples, which was used for isolation. These pups were fasted 24 hours prior challenge, this method supported with method of [8] who done the experiment by feeding intestinal scrapings of infected dogs to fasted susceptible dogs. Clinical observations set out in (table 1 and 2). The animals were showed no change in behavior at first three days just change in consistency of feces in three dogs at 2nd day to mucoid diarrhea and gradually converted to severe yellow gray mucoid feces and last day contain blood. At 3rd day check the feces in at the floor by rapid test kit for the detection of shedding the virus and appearance of the positive results. At 4th day, every animal examined separately from each other and which result only three animals, which gave positive result at varies degree of severity.

At same time checking of total and differential count of WBCs were done , showed

significant increase in both

counts may be due to mixed infection of dogs because get it from field and may be pickup different types of infection make the response of body to all infections leukocytosis instead of leucopenia, cooperate the investigation of [10] who recorded that the leukocytosis occur due to response to complicating secondary bacterial infection. However, after 4th day these three animals begin to increase severity of signs there is poor condition, mucoid diarrhea, rough coat, sunken eyes and reduced appetite. From sixth day, the dehydration appeared and gradually developed until loss of elasticity. At ninth day, there is one of these three dogs died followed by death another two dogs with very poor condition. These results supported with [11] who recorded that the peak of viral shedding between 4

7 day post-inoculation.

All these results are in line with [12] , who records same observations when using of CPV2c in challenge instead of fecal suspension. However, the last two dogs still negative and healthy with rough coat and respiratory signs and one of them become weak positive at day 11 and then die at 14 and another become loss of appetite and poor condition and

sunken eyes and positive at day 15 and die at day 19 (table No1). Compared with control two pups which were remain normal till last day. Table (1). Results of experimental challenge of CPV orally. Days post inoculation (first group) No.

1

2

3*

1

N

N

N

2

N

N

N

3

N

N

N

4

5

N

N

N

N

N

N

4 ++ RC, MD + Same 1 ++ Same 1 N

N

N=Normal,

5 +++ Same

6 RA, SE, DN

7

8

9

10

11

12

13

14

Same

Same

PC

D

-

-

-

Normal TC, DC

++ Same

Same 1

Same

Same

D

-

-

-

-

Normal TC, DC

Same 2

Same 1

Same

Same

Same

D

-

-

-

Slight lymphopenia

N

+ RC, SE, RA

Same

Same

PC, BD

D, N TC, DC

N

RC, SE, RA

Same

++ Still live till 19 and die with PC

N

N

N

N

PC=Poor

N

N

N

N

Condition,

N

N

DN=Dehydration,

D=Death,

V=Vomiting,* Start viral shedding by rapid test, SE=Sunken eyes, BD=Bloody Diarrhea, MD=Mucoid Diarrhea, TC=Total Count, DC=Differential Count, RC=Rough Coat, RA=Reduced Appetite,! =Severe drop of total and differential WBCs count, + less severe in rapid test, +++ more severe.

The second group less age than first group but challenged after

I/M

injection

of

leptospiral

vaccine.

Clinical

investigations appeared early at 24 hour when the first dog become vomiting overnight and die due to dehydration and when examined of oral swab find of positive result. The

other animal, which become vomits with bloody diarrhea and die at 3rd day, at necropsy there is fibrin on heart muscle with congestion of lungs and intestine with signs of dehydration figure (1). The other two dogs show healthy but with bloody diarrhea and appeared positive result in rapid test at 5th day but die at 7 and 8 day respectively. The last dog, which resists infection in spit of positive result and bloody diarrhea, poor condition, rough coat and damage of one eye, then die at day 15. Moreover, all animals

in

this

group

showed

of

leukopenia

and

lymphopenia and neutropenia, because of these animals more susceptible to infection due to its age more young and more stressed than first group. This in line with [9] who recorded that enhancement of severity of parvovirus disease has reported in dogs receiving commercial distemper, hepatitis, leptospirosis vaccine several days prior to experimental challenge with virulent CPV2 due to immune suppression (table No.2).

Table (2): Results of the experimental challenge of CPV. Days post inoculation (second group) No .

1

2

3

4

5

6

7

8

9

1 0

1 1

12

13

1

D!

-

-

-

-

-

-

-

-

-

-

-

-

2

PC , RA

PC, RA , V, BD

D !

-

-

-

-

-

-

-

-

-

-

3

N

N

N

BD, RA, PC!

+++ Sam e

Sam e

D

-

-

-

-

-

-

4

N

N

N

Sam e 3!

Sam e3

Sam e3

DN ,V

D

-

-

-

-

-

N

BD , SE, DN

+ +

5

N

N

N=Normal,

N

N

N!

PC=Poor

N

Condition,

N

N

N

N

DN=Dehydration,

14 TC 1200 C/d1 TC 1350 C/d1 TC 2200 C/d1 TC 1290 C/d1 Die at day 15 with ocular damag e TC 3200 C/d1

D=Death,

V=Vomiting, SE=Sunken eyes, BD=Bloody Diarrhea, MD=Mucoid Diarrhea, TC=Total Count, DC=Differential Count, RC=Rough Coat, RA=Reduced Appetite,! =Severe drop of total and differential WBCs count

Figure (1). ). Fibrin on heart muscle with congestion of lungs were showed in pups at experimental infection .

Ac knowledge ment I preciate the efforts of all owners and friends at veterinary medicine college Baghdad university university .

References 1. Reed AP, Jones EV, Miller TJ. Nucleotide sequence and 1. genome

organization

of

canine

parvovirus.(

1988 1988).

J

Virol.;62:266 76. Virol.;62:266 2. Parrish CR, Carmichael LE. Antigenic structure and variation 2. of canine parvovirus type-2, type , feline panleukopenia panleukopenia virus, and mink enteritis virus.( 1983 ).J Virol.;129:401 Virol.;129:401 14. 3 Ettinger, S.J. and Feldman, E.C. (1995 3(1995). ). "Textbook of Veterinary Internal Medicine (4 ( th Ed.). W.B. Saunders Company Company. 4 Jones, T.C.; Hunt, R.D. and King, N.W. (1997). 4( ). Veterinary Pathology. Blackwell Publishing. Pathology.

5- Schatzberg, S.J.; Haley I.; Bar1, A. and de Lahunta K. (2002). Polymerase chain reaction amplification of parvovirus DNA from the brain of dogs and cats with cerebellar hypoplasia. ACVIM 2002, Cornell University Hospital for Animals, Ithaca, NY; College of Vet. Med. University of Missouri, Columbia, MO; Vancouver, British Columbia, Canada. 6- Greene, C.E. (1998). "Infectious Disease of Dog and Cat". 2nd Ed. Saunders, Philadelphia, PA. 7- Aiello, SE.;Mays, A.; Anderson, D.; Amstutz, H. (2006). Merck Veterinary Manual (2006). "Canine Parvovirus". 50th Ed. Merck and Co., Inc., NJ, USA. 8- Carman, S. and Povey, C. (1982). Successful experimental challenge of dogs with canine parvovirus 2. Can. J. Comp. Med., 46: 33-38. 9- Potgieter, L.N.D.; Jones, J.B. and Patton, C.S. (1981). Experimental parvovirus infection in dogs. Can. J. Comp. Med., 45: 212-216. 10- Boosinger, T.R.; Rebar, A.H. and DeNicola, D.B. (1982). Bone marrow alterations associated with canine parvo viral enteritis. Vet. Pathol., 19: 558-561. 11- Greene, C.E. (1984). "Clinical Microbiology and Infectious Disease of Dog and Cat". 1st Ed. Saunders, Philadelphia, PA. 12- Spibey, N.; Greenwood, N.M.; Sutton, D.; Chalmers, W.S.K. and Tarpey, I. (2008). Canine parvovirus type 2 vaccines protects against virulent challenge with type 2c virus. Vet. Microbiol., 128: 48-55.

Histopathological and Biolchemical studyto effect of codeineparacetamolin Sprague Dawley Rats

Prof.Dr. Adel J. Hussein .. Department of Anatomy& histology, College of Veterinary Medicine,University of Basra / / Aseel Kamel Hameed Department of Basic Science, College of Dentistry, University of Basra / / S.K.Majeed Department of Pathology & Poultry diseases, College of Veterinary Medicine, University of Basra / / [email protected]

ABSTRACT This study performed in twenty four male Sprague Dawley rats for (90) days which divided randomly into four equal groups. Group (1): received normal saline daily. Group (2): received orally codeine-paracetamol(40/2500 mg / kg b.w) daily. Group (3): received orally codeine-paracetamol (80/5000 mg / kg b.w)daily. Group (4): received orally codeine- paracetamol (160 /10000 mg / kg b.w) daily. After end of day (90) of study all animals were sacrificed to do the histopathological , histochemical and ultrastructure examinations. . The histopathological investigation of liver showed centrolobular necrosis, dilation of sinusoids, vaculation of hepatocytes and septal fibrosis of liver , these changes are appeared mild in group (2), moderated in group (3) and sever in group (4). The histochemical study of poly saccharides(glycogen) of liver showed decrease of poly saccharides composition in mild zonal area and near the central vein, those changes were more severe in (H.D).While the histochemical study of lipid showed increase of lipid composition in hepatocytes near the central vein and those lipid compositions increased when the dose increased. The electron microscope study of liver showed mild to moderate swelling of mitochondria and dilation of smooth endoplasmic reticulum in both (L.D) and(I.D) while (H.D) showed severe swelling of mitochondria and dilatation with proliferation of smoth endoplasmic reticulum. INTRODUCTION Paracetamol (acetaminophen) is a widely used analgesic and antipyretic agent for the relief of fever, headaches, minor pains, it is a major ingredient in numerous cold and flu

remedies; in combination with non-steroidal antiinflammatory drugs and opioid analgesics (1). Paracetamol is used also in the management of severe pain such as post operative pain; its overdose produces hepatic necrosis and renal failure due to increases in lipid peroxide levels and depletion of glutathione (2). Paracetamol was the fourth most common cause of death following self-poisoning in the United Kingdom in 1989 (3). The narrow margin between therapeutic and toxic doses, that reveals when ingestion of (10-15grams) of paracetamol by adults may cause severe hepato-cellular necrosis and less often renal tubular necrosis (4). Codeine phosphate is predominant alkaloid opium, it is considered as a pro-drug, metabolized to active compounds of morphine and codeine-6-glucoronide (5,6). Codeine is a classic analgesic cause nephropathy is characterized by renal papillary necrosis and papillary calcifications(7).On other hand the relative contribution of the opioid to the hepatotoxicity is unknown; also codeine distributed in the lung, liver, kidney, and spleen, it has pharmacologic effects on the central nervous system (e.g. analgesia, drowsiness, mood changes, respiratory depression, nausea, and dysfunction of the endocrine and autonomic nervous systems) and the gastrointestinal tract like decreased gastrointestinal motility (8,9). The combinations of codeine with paracetamol produce a significant increase in analgesia compared with Paracetamol alone ; codeine causes less euphoria and sedation than morphine, but CNS depression and coma

occur in case of overdose (10,11). The synergistic effect of paracetamol-codeine is possible via a pharmacokinetic interaction between the two classes of drugs, NSAIDs may decrease the renal excretion of the pharmacologically active metabolite of morphine (morphine-6-glucuronide), also codeine, a centrally-acting opioid, can have additive analgesic effects when combined with peripherally acting agents (12,13). MATERIALS AND METHODS Twenty fourmale Sprague Dawleyrats were used to do this study, their weights were ranged (150-200) grams. They were taken from animal house of Veterinary Medicine college at University of Kufa, then they were housed in laboratory animal house incollege of Veterinary Medicine at University of Basrah, with (25±2?C) temperature with lightness system was (10/14) hours darkness/lightness and a mechanical ventilations was used to control of the suitable humidity. They were kept for adaptation in these environments for (14) days before the study. These animals were divided randomly in to four groups and put in specific plastic cages. Each group of those rats were put cages, in each cage, there were six animals per each group to avoid crowding and nutrient a pellet, and water until the end of the study.Also the cages were made from a plastic containing hard-wood chip as bedding. The bedding was changed continuously to ensure a clean environment.Also the animals weighing in day zero, 30th , 60th and lastly in day 90th.

DETERMINATION OF MAXIMUM TOXIC DOSE In this experiment, we started with normal human tablet contain (500mg of paracetamol / 8 mg of codeine) brought from (Bristol pharmaceutical company / U.K), then gave orally for two rats then started to increase the dose until we reached to a dose of six tablets which gave us a clinical signs of the maximum toxic dose like drowsiness, non active , arching back, piloeraction, sunken of eyes and pin point of the pupil. At this point we decided that will be the maximum toxic dose is (15000 paracetamol / 240 mg of codeine / kg bw).Then we decided that the minimum toxic dose is the high dose (group 4) will be four tablets (10000 mg of paracetamol /160 mg of codeine /kg bw) and the intermediate dose (group 3) is two tablets (5000 mg of paracetamol / 80 mg of codeine /kg bw) while the low dose (group 2) is one tablet (2500mg of paracetamol/ 40mg of codeine /kg bw) (14). EXPERMENTAL DESIGN This experiment was divided into four groups each group contains six male rats as the following: Group (1): It received normal saline oral doses daily for (90) days which served as control group. Group (2): It received (40mg of codeine/2500mg of paracetamol/ kg bw) oral doses daily for (90) days which served as low dose group (L.D).

Group (3): It received (80 mg of codeine/5000mg of paracetamol / kg bw) oral doses daily for (90) days which served as intermediate dose group(I.D). Group (4):It received (160 mg of codeine/10000 mg of paracetamol/ kg bw) oral doses daily for (90) days which served as high dose group(H.D). COLLECTION OF SPECIMENS At the end of experiment (90) days, the rats were generally anaesthetized by inhalation of Chloroform and then sacrificed. Blood samples were collected directly from the heart by the use of disposable syringes of (5) ml capacity, then was poured into test tubes free from anticoagulant to isolate blood serum and allowed to clot at room temperature and then centrifuged at rate (3000 rotation/minute)for (5) minutes to isolate blood serum and froze at (-20C ) to estimate the biochemical parameters . After the collection of blood samples from the animals, the gross examination was done for all animals, the macroscopic appearance was recorded to detect any abnormal gross change in the internal organs, including location, color, size, shape, consistency and appearance of cut section, then the organs put in the formalin (10%) for fixation.

PREPERATION OF HISTOLOGICAL SECTIONS According to (15,16) the preparation of histological sections include:iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii Tissue samples were obtained from the internal organs like (Brain, liver and kidney), these specimens were fixed at (10%) formalin immediately after removal from the body, then washing the specimens with running water and dehydration in serial alcohol concentrations (70, 80, 90, 100 %).After that cleared in two stages of xylene, and infiltrated with paraffin at (56-57°C), then embedded in paraffin followed block making, and a thin tissue section cut at (6 µm) with rotary microtome, after that the section mounted on the glass slides, attached to the slide it's surface smeared by small drops of Mayer's albumin, then the slides were put on hot plate at (45C ) for (24) hours, finally the specimens were stained with hematoxylin eosin, then examined by light microscope to detect any histopathological changes. BIOCHEMICAL ANALYSIS Biochemical analysis was measured by using kits which include: 1. Liver function tests like (Aspartate aminotransferase (AST)

,

Alanine

aminotransferase

phosphatase (ALP), and bilirubin. 2. Kidney function test (creatinine).

(ALT)

,

Alkaline

STATISTICAL ANALYSIS The results were analyzed by ANOVA and exposed to independent T-test by using SPSS (special program for statistical system) version 14.0. A value of (P 0.05) was accepted as being statistically significant. RESULTS The statistical analysis result of effects of codeineparacetamol toxicity in body weight showedthere was a significant (P 0.05) changes between all treated groups when compared with control group in day (90) of treatment which especially there was a significant (P 0.05) decrease in body weight of high dose treated group (H.D) as shown in table (1). The histological examination of liver of control group showed normal liver architecture which consist of normal hepatocytes, central vein and portal areas as in figure (1). The histological study of liver of low dose group (L.D) revealed periportal fibrosis, septal fibrosis, vaculation of hepatocytes, dilation of sinusoids and mild centrilobular enlargement of hepatocytes as in figure (2). The intermediated dose group (I.D) revealed centrolobular enlargement of hepatocytes, septal fibrosis, vaculation of centrolobular region,dilation of sinusoids and mid zonal hepatocytes as shown in figure (3).While the high dose group (H.D) revealed severe centrolobular necrosis, congestion of sinusoids, hemorrhage, early septal fibrosis, diffuse enlargement of hepatocytes as seen in figure (4).

The histological results of kidney of control group revealed normal structures of kidney which contain normal glomeruli, proximal and distal convoluted tubules as shown in figure (5). The histological study of kidney of low dose group (L.D) and intermediate dose group (I.D) revealed mild to moderate vaculation of mesengial cells and vaculation of proximal convoluted tubules as shown in figure (6,7). The histological study of kidney of high dose group (H.D) revealed necrosis of proximal convoluted tubules, vaculation of mesengial glomerular cells, vaculation of proximal convoluted tubules and dilatation of proximal convoluted tubules as shown in figure (8). The histological study ofbrain of control revealed normal structures of nervous tissues which contain normal neurons (cell body and axon) , glial cells, astrocytes and oligodendritic cells as shown in figure (9). The histological examinations of brain of low dose group (L.D) and intermediate dose groups (I.D) showed mild vaculation of neurons as in figure (10) and(11) respectively.While the histological study of brain of high dose group (H.D) revealed vaculation of neurons, vaculation of oligodendritic cells and vaculation of granular layer cells as shown in figure (12). The statistical analysis results of codeineparacetamoltoxicity in the levels of serum enzymes showed there were non significant (P 0.05) differences between

intermediated dose group (I.D) and low dose group (L.D) after (90) days of treatment as in table (2).While there was a significant (P 0.05) increase in the level of total serum bilirubin (TSB) of the high dose group (H.D) when compared with control group. There was non significant (P 0.05) differences in the level of serum direct bilirubin (D.B) between all treated groups when compared with control group after (90) days of treatment as in table (2). There were a significant (P 0.05) increase in the levels of serum (AST), (ALT), (ALP) and creatinine among treated groups when compared with control group after (90) days of treatment as shown in table (2).i Table (1): Effect of codeine- paracetamol administration in body weight (grams). Groups

Day zero

Day 30

Day 60

Day 90

G1: Control

185 ± 7.63 a

203 ± 4.96 a

G2: L.D

184.1 ± 6.11 a 183.3 ± 6.66 a 182.5 ± 7.27 a

194.1 ± 6.11 a 193.3 ± 6.28 a 192.5 ± 5.28 a 187.1 ± 6.37 a

214.3 ± 4.98 a 212.1 ± 4.79 c 204.5 ± 3.6 a

G3: I.D

G4: H.D

200.8 ± 5.54 a 199.3 ± 5.05 a 189.0 ± 6.23 a

190.5 ± 5.64 b

The mean difference is significant at the (P 0.05) level. The symbol latters (a,b,c) means a significant difference among the groups.

Table (2): Effect of codeine- paracetamol administration in serum liver function test and creatinine levels: Groups

(G1) Control (G2) (L.D) G3

TSB

D.B

AST

ALT

ALP

Creatinine

(mg/dl)

(mg/dl)

(U/L)

(U/L)

(U/L)

(mg/dl)

0.13±0.02 0.01±0.001 a

a

0.15±0.02 0.02±0.003 a

a

0.13±0.02 0.02±0.003

(I.D)

a

a

G4

0.25 ±

0.01 ±

(H.D)

0.04 b

89±0.9 a

a

a

a

115±1.5

76.3±1.2

31.8±0.8

0.2±0.03

b

d

d

c

133±1.4

98.8±2.5

42.6±0.8

0.3±0.02

b

c

c

b

57.3±0.4

0.2±0.01

b

b

163.8 132±11.9 ±1.3

0.002 a

58.3±0.8 25.3±1.02 0.15±0.02

b b

The mean difference is significant at the (P 0.05) level. The symbol latters means a significant differences among the groups.



B C

A

Figure (1):Transverse section through the Liver of control group(H&E stain, 400X). A. Central vein B. Hepatocytes C. Sinusoids. A. . B. . C. .

A

B

Figure (2):Transverse section through the Liver of (L.D) group(H&E stain ,400X)A. Vaculation of hepatocytes. B. Dilatation of sinusoids.

46



A

B

Figure (3): Transverse section through the Liver of (I.D) group (H&E stain, 400X). A. Vaculation of hepatocytes. B. Dilated sinusoids.

B A

Figure (4): Transverse section through the Liver of (H.D) group (H&E stain, 400X). A. Centrilobular necrosis. B. Hemorrhage. . 47



A B

Figure (5): Transverse section through the kidney of control group ( H&E stain, 400X). A. Glomeruli. B. Renal convoluted tubules.

A

B

Figure (6): Transverse section through the Kidney of low dose (LD) group (H&E stain, 400X). A. Vaculation of mesengial glomerular cells. B. Vaculation of proximal convoluted tubules. 48



A

B

Figure (7): Transverse section through the Kidney of intermediate dose (ID) (H&E stain, 400X). A. Vaculation of mesengial glomerular cells. B. Vaculation of proximal convoluted tubules.

D B A C

Figure (8): Transverse section through the Kidney of high dose group (HD) (H&E stain, 400X) A. Vaculation of mesengial glomerular cells. B. Vaculation of proximal convoluted tubules. C. Necrosis of proximal convoluted tubules. D. Dilatation of convoluted tubules. 49



D

B

C

A Figure (9):Transverse section through the brain of control group ( H&E stain, 400X) A. Neurons. B. Axons. C. Astrocytes. D. Oligodendrocytes.

A

Figure (10): Transverse section through the Brain of low dose (L.D) group ( H&E stain, 400X). A. Vaculation of neurons.

51



A

Figure (11): Transverse section through the Brain of Intermediate dose (I.D) group (H&E stain, 400X). A. Vaculation of neurons.

A

B

Figure (12): Transverse section through the Brain of high dose (H.D) group (H&E stain, 400X). A. Severe vaculation of neurons. B. Congested blood vessels (vein).

51



DISCUSSION The statistical analysis study of codeine-paracetamol in animals body weight showed there was a significant (P≤0.05) decrease in body weight of high dose treated group (H.D) after (90) days of treatment as in table (1).These could be due to a compromised nutritional status of the rats consequent on gastrointestinal tract derangement, also the loss of appetite combined with drowsiness due to insufficient intake of opium could lead to weight lossas same with (17,18). This present study is in agreement with (19,20) whomentioned that paracetamol treated rats had a significantbody weight loses (P