Clinico-Therapeutics and Immunological Studies on Strangles in Donkeys

4. أ . د / نبيل محمد عبدالحميد بكير أستاذ الأمراض المعديه المساعد كلية الطب البيطرى- جامعة دمنهور ومدرب معتمد TOT

5. Clinico-Therapeutics and Immunological Studies on Strangles in DonkeysNabilM.Bakir,NohaA.Beder and Hassan A.Hammoda

6. Introduction

7. Egypt is an agricultural based country and livestock plays an important role in the nation’s economy. • The donkey in fact plays an important role in the provision of energy for agricultural production • there are 1 million working donkeys in Egypt These donkeys are often the owners’ only source of income and transport, and are extremely important to an entire family’s survival. • Even today,thestoic,hard working donkey is often misunderstood by its owner and ,unfortunately,often by veterinarians worldwide. • In third world countries,because of the poverty of the owners,vets are rarely called,and in more prosperous countries where veterinary attention is given there is tendency for vets to treat the donkey as asmall horse,

8. Strangles is one of the most important bacterial diseases,which affects horses, donkeys and mule. strangles is caused by Streptococcus equi subspecies equi, a Gram positive, beta hemolytic organismand has been reported from different countries (Timoney,1999; Chanter et al., 2000; Newton et al., 2000; Khoo et al., 2011; Erol et al., 2012; Senthil et al., 2014). • It affects animals of all age groups. • The morbidity may be up to 100%.

9. About 10% of the affected animals may die from disseminated abscessation or purpurahaemorrhagica (Chanter et al., 2000). • The infection is transmitted through direct contact with nasal discharge or pus from infected donkeys or fomites. Animal to animal transmission involves direct face- to- face contact or exposure to contaminated feed, water, veterinary instruments, grooming kit, beside twitches or bridle bites (Radostits et al., 2000). • Isolation of the organisms still remains the gold standard for the diagnosis of strangles

10. Depression of immune response is commonly found in various diseases, including • malaria ,trypanosomiasis ,babesiosis, • various microbial agents • mycotoxicosis, • misuse of antibiotics • other factors which interfere with the normal immune response. (Greenwood et al.,1971),

11. Immunomodulators are substances that act on the immune system and have the capacity to increase or decrease the immune response,immunopotentiators act in combination with antigens stimulation and augment the ongoing respose(Mulcahy and Quinn,1986). • Immunostimulants are used in attempt to counteract the effects of such environmental immunosuppresion factors. • various immunostimulants have been reported in the literature,including Bacillus and Guerin(BCG) (Fudenberg et al.,1977;Barakat et al.,1981and Osman et al.,1987).

12. So,the present study wase designed to investigate: 1- the effect of BCG as an immunostimulant when administered to donkeys. 2- The clinical and postmortem signs of naturally infected donkeys with strangles. 3- The effect of the disease on blood picture of infected donkeys. 4-The non-specific immunostimulant effect of BCG through cell mediated immune response on prevalence of infection and cure rate 5-The effect of the different treatment against strangles.

13. MATERIAL AND METHODS

14. 1-Animals: • 40 donkes of both sexes,.with age from one day -7 year 20 donkeys were vaccinated with BCG vaccine and the rest of the animals non-vaccinated, 2-Methods : • Bacillus calamite and Guerin ( BCG). • Blood sample and serum sample • Nasal swabs and pus sample (Quinn et al. (2007). • - Haematological studies:(Schalm et al.,975 and Feldman et al., 2000) - Measurment of serum proteins,albumine and globulin (A/G ratio)(Mc Beath et al.,1971 and Khan et al., 2010). - Phagocytic index and phagocytic activity (Kawahara et al.,1991). - Lymphocyte transformation index and cell mediated((Rai El-Balhaa et al.,1985) - Treatment: 1.penicillin and streptomycin 2.Oxytetracycline all animals administered haematinic with anti-inflammatory in diseased one -Statistical analysis

15. Result

16. Clinical finding • A marked variation in the clinical signs was recorded in the clinically affected donkeys according to the stage of infection. • During the early stage,affected donkeys showed complete anorexia,depression,fever(39.5-40c,103-105F),serous nasal discharge • ,In some cases in late stage purulent,severepharyngitis and laryngitis,in some cases especially in youg age unable to swallow and regurgitate milk from nostrils,moist cough with pain and easily stimulated by compression of the pharynix ,other donkeys during the later stages showing characteristic abscesses in the lymphnodes of the throat region which is hot,painfull and swollen and accompanied by snoring sound and other cases discharging pus from affected lymphenodes in throat region, • Post mortum lesions in complicated dead cases reveals extensive suppuration in internal organs as liver,spleen,lungs and pleura.

17. Table 1 : Clinical details of the Steptococcusequi positive donkeys:

18. Microbiological examination • The clinical details of the S. equi positive animals are presented in (Table 1) : • Of the 40 samples, seven(9 ) ( 5 nasal swabs,2 nasal from subclinical cases and 2 pus ) samples were positive for S.equi. • Gram’s stained smears revealed Gram positive cocci which were arranged in pairs and short chains . • Clinical specimens on blood agar showed small, circular,translucent,glistening colonies with beta haemolysis. • There was no growth on Mc Conkey’s medium. • The isolates grew well on Edward medium at 37Co.

19. Biochemical tests of Streptococcus equi isolates: • The fermentation tests as showed in (Table 2) revealed that : • the isolates were positive for maltose and negative for lactose, sorbitol and trehalose • All isolates were also found negative for catalase and oxidase. • Based on cultural examination,microscopic morphology and biochemical reaction, all the nine isolates were identified as S. equi

20. Table2 : Biochemical tests of Streptococcus equi isolates obtained from nine (9 )donkeys:

21. .Haematological changes   • - Haematological parameters as showed in (Table 3) found that: • there was significant increase in total leucocytic count, neutrophils, lymphocytes count and percent in vaccinated animals compared to contact non vaccinated animals(P < 0.01). • There was asignificant increase in total protein,albumin ,globulin(A/G ratio) in vaccinated clinically normal donkeys compared with non vaccinated clinically normal donkeys and found non significant difference in total RBCs count,Hb% and PCV between two groups

22. Table 3 Haematological parameters of vaccinated and non vaccinated clinically normal donkeys

23. Haematological changes   • Values with different superscripts in the same row are significantly different from each other (P < 0.01). • Each value represents as mean ± SE *RBCs=mean Red blood Cell count **PCV = mean Packed Cell Volume % *** Hb = mean Haemoglobin % ****WB = mean White blood cell concentration *****A/G rtio = Albumine /Globulin ratio

24. Table 4 Effect of strangles on blood picture,A/G ratio at the peak of infection,during and after treatment

25. Table 4 Effect of strangles on blood picture,A/G ratio at the peak of infection,during and after treatment

26. .Haematological changes   • -Different treatment in animals and haematological parameters as showed in (Table 4)found that: • The temperature,microrganim return to normal and negative simulatenously within 1-2 weeks after treatment with procaine penicillin combined with streptomycin while oxytetracycline showed delayed results. In infected donkeys, there was a significant increase in the total Wbcs count, neutrophil,lymphocyte concentration (p<0.01) while Hb%,PCV and total RBCs were non significantly increased in infected groups (p<0.01) compared to non infected. There was significant increase in total leucocytic count, neutrophils, lymphocytes, in infected animals treated with penicillin/streptomycin and oxytetracyclin BCG vaccinated group compared to penicillin/streptomycin and oxytetracyclin non vaccinated group - There was non significant difference within group befor and after infection, -- Significant difference in total protein,albumin and globulin between BCG vaccinated and non vaccinated and A/G ratio increase in vaccinated group.

27. Table 5 : Cell mediated immune responses of donkeys injected with BCG as measured by lymphocytic blastogenesis::and phagocytic indexMeans within the same column carrying different letters are significantly different at(p <0.05). N.s.=Non significance **=Significant at (p<0.01)

28. . Effect of BCG on immunity parameters and the prevalence and cure rate of strangles: • The result showed that BCG vaccinated donkeys gave highest lymphocyte transformation(69.66-55.23),phagocytic index(10-7.5) • while non vaccinated donkeys showed (49.01-42.00).(6.66-4.54) at 7and 15 days after injection simultaneously (table 5)

29. Table 6 Effect of BCG vaccine on the prevalence rate of stranglesprevalence of infection in vaccinated donkeys 10% while in non vaccinated 26.84%(table 6) and currency in BCG vaccinated donkeys was 98% while in non vaccinated 92.63%.as in table(7).

30. Table6 Efficacy of drugs on BCG vaccinated diseased donkey and Non-vaccinated diseased donkey

31. Discussion

32. Strangles is a highly contagious disease of equines,which involves the upper respiratory tract.The disease can occur in sporadic as well as in epidemic form . • However, the laboratory confirmation is imperative to establish an unequivocal diagnosis of strangles.Haryana Vet. ( 2015) In the present study, the clinical and microbiological findings conclusively proved that seven(7) of the 40 donkeys were suffering from strangles and two(2) of the clinically normal was positive microbiology to strept.equi . • In the absence of PCR, repeated culture of the nasopharyngeal swabs can be useful to detect the asymptomatic carriers of S. equi following outbreaks of strangles.

33. - The isolation of S. equi from the nasopharyngeal swabs of horses has also been reported by Senthil et al. (2014). These workers also recovered S. equi from the apparently healthy horses.Due to lack of PCR facility in our laboratory, we could not process any sample/isolate by PCR. It is possible that by employing PCR, we might have detected more animals positive for S. equi. • Newton and co-workers (1997) from USA reported that clinically healthy long term carriers of S. equi present a serious risk of spreading strangles, and therefore, repeated nasopharyngeal examination is imperative to detect the carriers. This study found that two subclinical cases identified by microbial isolation from 33 clinically normal donkeys which supported by(Animal Health Trust ) which sayed that animals carrying strept.equi can be difficult to detect using culture test and must be confirmed by guttural pouch endoscopy

34. early treatment with antibiotics may be helpfull but not always usefull in late stage as they cannot penetrate the center of an abscess where there is no blood supply , • The significant increase in lymphocyte transformation and phagocytic index can amplifier by Timoney et al.,2008;Flock et al.,2012 there reported that during infection and to elicit strong persistent serum and mucosal antibody resposes,aspecific antibodies were shown to neutralize antiphagocytic activity

35. Measures such as: • immediate isolation of clinically suspected animals, • administration of penicillin and streptomycin to the infected and in-contact donkeys, • thorough disinfection of the stables and equipments, • avoiding overcrowd and mixing of different age groups • prohibition on communal drinking water source • were undertaken so as to reduce the incidence of strangles in equine including donkeys. • All these measures have been reported to prevent the spread of the disease (Sweeney et al., 2005). Further studies on epidemiology of strangles in donkeys are needed in this region. • This study thus revealed the presence of S. equi in donkeys in Egypt.

36. The infected donkeys reflect decrease of immune resonse ,similar observation were recorded by (James et al.,1981),who observed anon-specific decrease in immunoglobulins(IgM and IgG) leading to immunosuppresion during acute infection , • this study reflected the increasing immune resonce of vaccinated donkeys with BCG vaccine In agreement with( Salma et al.,1997) who hound that BCG vaccinated lamb showed higher stimulated cell mediated immune response.this ,also (Aronson et.al.,2004) foud that although BCG vaccine shown protection against disseminated form of tuberculosis has also shown some protection against leprosy and certain form of cancer through BCG activate APCs via PAMPs that interacts with TLRs which initiate inflammatory cascade therby recruiting inflammatory cells to site of infection and providing maturation signals for neutrophils,macrophages and dendritic cells and leading to restriction of infection at the initial site and

37. also(Tizard 1992) explained by the fact that BCG bacilli contain acompound called Muramledipeptide which act on macrophages to produce Interleukin-1 ,which inturn stimulate cells of immune system and also promote antigen processing and T-cell mediated activity and all of them explained why BCG vaccinated donkeys showed • increase parameters of immunity, • lowering prevalence rate of infection • ,high cure rate compared with non vaccinated. • On the other hand these result disagree with those reported by (Magid and Kaneno,2000)they found that ,propionibacteria injected animals showed higher cell mediated immune response than BCG injected ones and (Calhoun et al.,1980),found that BCG and propionibacteria increase immune resonce in the same degree

38. Conclussion

39. 1-Non specific stimulation of defence mechanism is of great importance for prevention of all sorts of immunosuppressive influence in animal breeding through cell mediated immune response , prevalence of infection and cure rate and should be applied in vaccination programme of donkeys . 2- A/G act as an index to the activity of the immune system 3-In the absence of PCR, repeated culture of the nasopharyngeal swabs can be useful to detect the asymptomatic carriers of S. equi following outbreaks of strangles in donkeys. 4-The procaine penicillin with streptomycin is adrug of choice to treatment of early strangles signs in donkey due to they have marked effect on micro-organism, upset signs and decreasing rate of complications.

40. مع تحياتى أ . د / نبيل محمد عبدالحميد بكير