Bacteria and fungi associated with abortion in sheep and goat in Menoufiea Governorate

By

Elham I.  Atwa^* and Flourage M. Rady^**

^*Bacteriology and ^**Mycology Department, Animal Health

Research Institute Shebin El- Kom-Menoufiea

SUMMARY

     A total of (120) samples of aborted foeti, vaginal discharage and Placenta were collected from (70) aborted ewes and (50) aborted she goats from Menoufiea Governorate for bacteriological and mycological examination. Swabs from stomach and intestinal contents of aborted foeti as well as liver , spleen, and lungs were collected. The bacteriological examination of aborted foeti, vaginal discharage and Placenta of aborted sheep revealed the isolation of Brucella melitensis, Campylobacter fetus subsp.fetus, Campylobacter fetus subsp.venerealis, Listeria monocytogens,  Salmonella typhimurium, Salmonella dublin, Escherichia coli and Staph. aureus with the incidences of 21.4%, 11.4%, 7.1%, 8.6%, 5.7%, 2.9%, 1.4% and 1.4%, respectively. While the bacteria isolated from aborted she goats were Brucella melitensis, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp.venerealis, Listeria monocytogens,   Salmonella typhimurium, Salmonella dublin, Staph. aureus and Escherichia coli with the incidences of 20%, 10%, 6%, 10%, 8%, 4%, 4% and 2%, respectively.         

     Mycological examination of aborted foeti, vaginal discharage and Placenta of aborted sheep revealed the isolation of Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Candida albicans , Candida krusei, Mucor spp. Abisidia spp. and Rhodotrula spp. with incidence of (12.9%,5.7%, 2.9%, 8.6%, 2.9%, 4.3%, 2.9% and 1.4%, respectively). While the fungi isolated from aborted she goats were Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Candida tropicalis, Candida albicans, Mucor spp., Rhizopus spp., Penicillium pp. and Fusarium spp. with incidence of (14%, 6%, 2%, 10%, 8%, 6%, 4%, 2% and 2%, respectively).

    The stomach contents of the aborted foeti of ewes and she goats were the most common seat for the isolation of bacteria and fungi which cause abortion

     In vitro the antibiogram test indicated that the different bacterial species were more sensitive to danofloxacin, gentamicin, erythromycin and amoxicllin & clavulanic acid. While the most fungal isolates were sensitive to clotrimazole and  miconazole. 

     PCR (Polymerase chain reaction assay) was a valuable tool for direct and rapid diagnosis of Brucella melitiensis andAspergillus fumigatus from aborted foeti specimens. The amplification of 169 and 792 bp fragments from the extracted DNA of Brucella melitensis, while 383 bp fragments from the extracted DNA of Aspergillus fumigatus were done.

INTRODUCTION

     Sheep and goat represent an important sources of meat and milk production as human consumption in Egypt. High need of animal protein in Egypt increases year by year .So to overcome the problem of this deficiency, the maintenance of good fertility in herds is important because the reproductive health of animals is related to the nutritional needs of human population from meat, milk and wool for manufacturing purposes.

     These large farms met various problem especially the abortion problem which is initiated through various causes. Abortion is caused by many factors as mechanicl , chemical, nutritional , bacterial and mycotic causes .

     Bacterial abortion caused by Brucella melitiensis , Campylobacter fetus , Listeria monocytogens, Salmonella spp., Escherichia coli, Leptospira, Staph. aureus, Streptococci, Corynebacterium pyogens and Chlamydia spp.       (Kholeaf et al., 1977 ,  Butachaiah and Khera ,1982 , Bajmocy et al., 1987, Plagemann, 1989 and Sargison et al.,2001).

      Mycotic abortion caused by Aspergillus spp. Candida spp. Rhodotorula spp. Absidia spp., Alternaria spp. and Mucor spp. ( Pal et al., 1985 , Pal, 1988 and Verma et al., 1999).

     Brucellosis is a zoonotic disease that cause abortion, fetus death and genital infections in animals and humans . The illness initially presents as fever  and may later affecting various organs and tissues ( Redkar et al., 2001).    Brucellosis is considered one of the major problem affecting sheep and goats, producing many economic losses due to abortion and infertality (Butachaiah and Khera, 1982 ). Sheep and goats are mainly affected by Brucella melitiensis ( Wilson and Miles, 1975).

     Vibronic abortion of sheep and goat are characterized by abortion during the last half of gestation period , the disease is extermely sporadic . The incidence of abortion in sheep and goat occur due to Campylobacter fetus may reach up to 70% ( Flaat and Roed, 1980, Bird et al., 1984 , Bajmocy et al., 1987 and Varga, 1990) . While listeria monocytogens is a public health concern and affect human whose immune system are inefficient, and in pregnant women cause infant death, meningitis and abortion. In infected sheep and goats, abortion occurred at early stages of pregnancy and stillborn or weak kids (Plagemann ,1989 ).

     Fungi are pathogenic to man and animals, and are able to grow saprobiologically. They produce serious disease symptome as inflammation of the genitalia especially endometritis with mucopurulent discharge. They may be responsible for causing infertility, and abortion (Ainsworth and Austwick, 1973). Mycotic abortin is caused by fungal infection of the genital tract by several moulds and yeasts(Kirkbride, 1990 and Knudtson and Kirkbride, 1992).Abortion usually occurred during the last trimester of pregnancy (Williams et al.,1977 and Corbel,1988).The fungus Aspergillus fumigatus and Aspergillus niger were isolated in pure culture from cases of abortion in ewe by several researchers as ( Siddique et al., 1976, Cuci, 1987 , Pal, 1988 , Vandyousefi & Zoghi 1988 and Patnaik et al., 1992).

     Mycotic abortion among ewes reflects its isolation for the first time in India. Aspergillus fumigatus from cases of metritis and abortion in cows (Pathak & Mittal, 1966 and Pal et al., 1985). Mycotoxins in the genital tract are spermicidal to spermatozoa, as documented by Saxena and Ishaque (1977).

      Identification of Brucella melitiensis andAspergillus fumigatus by isolation was time consuming and the cultures need to be handled with care because of the zoonotic potential. So PCR assay was used for confirmation of presumptive Brucella melitiensis and Aspergillus fumigatus isolates , allowing the rapid diagnosis and facilitated studies of microorganisms (Brieker and Halling 1994, Cetinkaya et al., 1999, Liliana et al., 2004, William et al., 2004 and David et al., 2005).

    The aim of this study is to prove the microbiological causes of sheep and goat abortion. This can be established through demonstration, isolation and identification of the bacterial and mycotic agents. On the other hand, their susceptibility to chemotherapeutic agents was done as an aid to overcome this problem and reduce losses. Also, using polymerase chain reaction (PCR) test to substitute the conventional cultural methods and rapid diagnosis of Brucella melitiensis and Aspergillus fumigatus. 

MATERIAL AND METHODS

Samples:

     120 samples of aborted foeti were obtained under complete aseptic condition from 70 ewes and 50 she goats for bacteriological and mycological examination. Swabs from all number of examined samples of stomach and intestinal contents of aborted foeti as well as liver , spleen , and lungs were collected in a separete sterile containers and were transported as quickly as possible to laboratory in ice box (Animal Health Research Institute – Shebin El-Kom ).

    Placenta and vaginal discharage were collected aseptically from aborted ewes and she goats by sterile cotton swabs and tranferred immediately to the laboratory , where they were examined bacteriologically and mycologically.

    All samples were obtained from aborted ewes and she goats from various private farms at El- Menoufiea Governorate .

Bacteriological examination:

     The collected samples including swabs from stomach (abomasal), intestinal contents, vaginal samples and placenta of the aborted foeti as well as internal organs were inoculated directly onto the Albimi agar plates, Cmpylobacter blood free selective agar , blood agar ,MacConkey^,s bile salt lactose agar, S.S. agar and  mannitol salt agar. The inoculated plates for the Albimi agar plates was incubated in aerobic condition in jars or incubator containing 5-10% CO₂ at 37^oC. Cmpylobacter blood free selective agar supplemented with antibiotics was incubated in microaerophilic condition of reduced 5% O₂ , 10% CO₂ using CO₂   generating kit and 85% N₂ in plastic anaerobic jar at 37 ^oC for 3- 4 days . Then suspected Brucella colonies were identified morphologically, staining reactions and biochemically according to Alton et al., (1975). While Campylobacter colonies were identified according to Skirrow and Benjamin, (1980)  and Prescott and Munroe, (1982).

    The inoculated plates for the last four media were incubated at 37^oC for 24-48 hours, then suspected colonies were picked up and streaked onto nutrient agar slant , incubated at 37^oC for 24 hours to obtain pure culture. Suspected colonies were identified morphologically, Gram^,s stain reactions and biochemically according to the Koneman et al., (1992) and Quinn et al., (2002).

Mycological examination:

     The collected samples,(swabs from stomach (abomasal), intestinal contents, vaginal samples and placenta of the aborted foeti as well as internal organs) were inoculated onto the surface of Sabouraud^,s dexrose agar (SDA) containing 0.05% chlooramphenicol, and Candida agar (CA), the spot inoculation method was followed to culture fungi . Plates were inoculated at 25^oC for a minimum period of 7 days . The inoculated plates were examined for fungal growth , texture, diffusable pigment and morphological descripiton according to             Raper and Fennel, (1965), Carter and Cole, (1990) and Koneman et al., (1992).

 Susceptability of isolates to chemotheraputic agents :

      The standardized disc agar diffusion method was applied on a pure subcultures to detect the drug of choice against different bacteria isolated strains accordind to Finegold and Martin (1982). The results were interpretated according to Koneman et al., (1992).

     While use of filter paper disks (3mm in diameter) were used for preparation of antimycotic disc by soaked disk in 1 ml of drug used and left to complete dryness. Disks from each drug were put in the plate and incubated at 25 ^oC for 2-3 days according to Colle et al., (1996). The results were interpretated according to  Rippon (1988).

Extraction of Brucella melitiensis DNA according to Sambrook et al., (1989) and William et al.,  (2004):

     Five ml of trypticase soy broth were inoculated with bacterial strains at 37^oC for 24-48 hours. Spin 1.5 ml of culture in microcentrifuge for 2 minutes until the compact pellet forms and the supernatant discarded. The pellet resuspended in 500ul of TE buffer (Tris-EDTA buffer).Then 50uL of 10% SDS (Sodium dodocyle sulphate) and 3mg/ml proteinase K to give final concentration of 100ug/ml proteinase K in 0.5% SDS then mixed and incubated for 1hour at 37 ^oC. Then add 100ul of 5M NaCL and mixed to remove cell wall debris, denaturated protein and polysaccharides complexes. While retaining of the nucelic in the solution. Add apprximately equal volume of chloroform/ isoamyl alcohol then mixed and spins 4 to 6 minutes in a microcentrifuge, the aqueous , viscous supernatant is put in microcentrifuge tube with equal volume phenol / chloroform / isoamyl alcohol, extracted thoroughly, and spin in a microcentrifuge for 5 minutes. The supernatant is put in fresh tube with 0.6 volume isopropanol was added to precipitate nucleic acid , the tube was shaked until the DNA precipitate, then pelleted by spinning at room temperature. DNA was washed with 70% ethanol to remove residual and respinned 5 minutes at room temperature repellet, then remove supernatant and dried pellet by lyophlizer. DNA pellet was redissolved in 100 ul of TE buffer.

Primers for Brucella melitiensis:

     Specific oligonuclotide multiplex primer assay designated by Brieker and Halling (1994) and Ewalt and Bricker (2000) as AMOS for rapid differenenation between (Abortus, Melitiensis, Ovis, Suis), The forward primer                                   forBrucella melitiensis was(5`-AAA-TCG-CGT-CCT-TGC-TGG-TCT-GA-3`). While the IS711 was used as a reverse primer (5`-TGC-CGA-TCA-CTT-AAG-GGC-CTT-CAT-3`). Specific primer forBrucella melitiensis was amplified fragment at:169bp and 792bp. The specific oligonuclotide primers were obtained from MWG Biotech AG and used as pooling primers for AMOS amplification.

Brucella melitiensis DNA amplification by PCR:              

     DNA samples were denaturated by boilling for 10 minutes before amplification and then quenched on ice. The PCR was performed according to Bricker and Halling(1994) in a touchdown thermocycler in a total reaction volume of 50 ul containing 60 mM tris- HCL (pH 9.0), 1.5 mM MgCL₂, 15mM (NH₄)₂SO₄, 250mM each of the four deoxynucleotide triphosphate, 0.2 mM of each primer, 1 unit of Taq polymerase and 200uL of extracted DNA. The PCR mixtures were over laid with 40uL paraffin oil and ampilified in DNA thermal cycler. Amplification was obtained with 35 cycles. Each cycle involved denaturation at 95^oC for 1.5 minutes, annealing at 55 ^oC for 2 minutes , and extension at 72 ^oC for 2 minutes. The final extension was performed at 72^oC for 5 minutes  .

     For the detection PCR products , a 10 ul of amplified DNA was examined by electrophoresis in 1.5% agarose gel, and visualized with ethidium bromide and UV light . Electrophoresis was carried out for 2 hours at 110 V.

Extraction of Aspergillus fumigatus DNA according to Liliana et al., (2004) and David et al., (2005):

     Aspergillus fumigatuswasgrown on 5 ml sabourad dextrose agar in 50 ml flask at 37 ^oC for 2 days and then left at 25 ^oC to sporulate until mature. The agar was overlaid with 10ml of sterile 0.1% Tween 20, then placed in rotary shaker for 10 minutes. The conidia and hyphal fragments of Aspergillus fumigatus was harvested and passed through 5um polycarbonate filter to remove hyphae.Stir bar was used to break hyphae by spinning on a stir plate.Hyphal fragments were pelleted by centrifugation at 3,200 x g for 15 minutes and resuspended in sterile water. The DNA was precipitated with isopropanol and sodium acetate , washed with 70% ethanol and resuspended in Tris-EDTA buffer. The extraction of DNA by (Ultra Clean soil DNA isolation kit) uses a bead matrix and lysis buffer to pulverize cells by horizontal shaking on a vortex mixer, followed by adsorpation of DNA to a spin filter, a wash step, and the dilution of DNA in  TE buffer.

Primers for Aspergillusfumigatus:

     Aspergillus fumigatus specific primer sequence were published by David et al., (2005) the forward primer for PCR was Fun-18S-995F (5`-CGA TYA GAT ACC GTY GTG TC-3`). While the Fun-18S-1217R was a reverse primer (5`-TGT CTG GAC CTG GTG AGT TT-3`). Specific primer for Aspergillus fumigatus was amplified fragment on :383bp. The specific oligonuclotide primers were obtained from  Invitrogen Corp., Carlsbad.

Aspergillusfumigatus DNA amplification by PCR:              

     2ul of diluted DNA samples were mixed with 20 mM tris- HCL, 50mM KCl, 1.5mM MgCL₂, 0.2mM each of the four deoxynucleotide triphosphate, 0.5 mM of each primer and 0.5 unit of Taq DNA polymerase ,in total volume of 20uL. PCR ampilification conditions were 5 minutes of denaturation at 96^oC, followed by 40 cycles of 94 ^oC for30 seconds, 58 ^oC for 30 seconds,and 72 ^oC for 30 seconds. The final extension step was performed at 72 ^oC for 15 minutes, (David et al., 2005).   

RESULTS

     Results in Table (1), show the bacterial and fungal isolates from aborted foeti, vaginal discharge and placenta of aborted ewe. Out of 70 samples, 42 samples (60%) were positive for bacteriological isolates, while 29 samples (41.4%) were positive for mycological isolates.

     The bacteria isolated from ewe aborted foeti were Brucella melitensis, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp.venerealis, Listeria monocytogens and Salmonella typhimurium with incidence of ( 10%, 7.1%, 4.3%, 4.3% and 2.9%, respectively). While the bacteriological isolation from vaginal discharge were Brucella melitensis, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp.venerealis, Listeria monocytogens,   Salmonella typhimurium, Salmonella dublin and Escherichia coli with incidence of ( 4.3%, 1.4%, 1.4%, 2.9%, 1.4%, 1.4%, and 1.4% respectively). The bacteriological isolation from placenta were Brucella melitensis, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp.venerealis, Listeria monocytogens, Salmonella typhimurium, Salmonella dublin and Staph. aureus with incidence of (7.1%, 2.9%,1.4%, 1.4%, 1.4%, 1.4% and 1.4%, respectively).

     The fungi isolated from ewe aborted foeti were Aspergillus fumigatus, Aspergillus niger, Candida albicans, Mucor spp. and Abisidia spp. with incidence of (8.6%,1.4%,4.3%, 2.9% and 1.4%, respectively).The fungi isolated from vaginal discharge were Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Candida albicans , Candida krusei, Mucor spp. Abisidia spp. and Rhodotrula spp. with incidence of (2.9%,1.4%, 1.4%, 2.9%, 1.4%, 1.4%, 1.4% and 1.4%, respectively). While the fungi isolated from placenta were Aspergillus fumigatus, Aspergillus niger, Candida albicans and Candida krusei with incidence of (1.4%, 1.4%, 1.4% and 1.4%, respectively).     

     In attempt to correlate the relation between various types of bacteria and fungi and their sites of positive aborted ewe foeti, the data obtained were recorded in Table (2).Out of 20 aborted ewe infected with Brucella melitensis, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp.venerealis, Listeria monocytogens and Salmonella typhimurium the organisms were present mainaly in the fourth stomach cotents with incidence of 100%  in all bacterial isolates, then marked drop in liver with an incidence of (85.7%, 80%,100%, 66.7%, and 100%, respectively), followed by spleen with an incidence of (85.7%, 60%, 66.7%, 66.7% and 50%, respectively).Also lung specimens were the least common seats of infection with an incidence of (71.4%, 60%, 66.7%, 0%, and 50%, respectively) .

     While Out of 14 aborted ewe infected with Aspergillus fumigatus, Aspergillus niger, Candida albicans, Mucor spp. and Abisidia spp.The fungi were present mainaly in the fourth stomach cotents with incidence of 100%  in all fungal isolates, then marked drop in lungs with an incidence of (100%, 50%, 66.7%, 100% and 100%, respectively), followed by liver with an incidence of (83.3%, 100%, 66.7%, 50% and 0%, respectively).Also spleen specimens were the least common seats of infection with an incidence of (66.7%, 50%, 33.3%, 0% and 0%, respectively) .

      Results in Table (3), show the bacterial and fungal isolates from aborted foeti, vaginal discharge and placenta of aborted goats.Out of 50 samples,32  samples(64%) were positive for bacteriological isolates, while 27 samples (54%) were positive for mycological isolates.

    The bacteria isolated from goat aborted foeti were Brucella melitensis, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp.venerealis, Listeria monocytogens, Salmonella typhimurium, Salmonella dublin and Staph. aureus with incidence of ( 12%, 6%, 2%, 4%, 4%, 2% and 2%, respectively). While the bacteriological isolation from vaginal discharge were Brucella melitensis, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp.venerealis, Listeria monocytogens, Salmonella typhimurium, Salmonella dublin and Escherichia coli with incidence of ( 4%, 2%, 2%, 2%, 2%, 2%, 2% and 2% respectively). The bacteriological isolation from placenta were Brucella melitensis, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp.venerealis, Listeria monocytogens, Salmonella typhimurium and Staph. aureus with incidence of (4%, 2%, 2%, 4%, 2% and 2%, respectively).

     The fungi isolated from ewe aborted foeti of goats were Aspergillus fumigatus, Aspergillus niger, Candida tropicalis, Candida albicans, Mucor spp. and Rhizopus spp. with incidence of (8%, 4%, 4%, 2%, 2% and 2%, respectively).The fungi isolated from vaginal discharge were Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Candida tropicalis, Candida albicans, Mucor spp., Rhizopus spp., Penicillium pp. and Fusarium spp. with incidence of (4%, 2%, 2%, 2%, 2%, 2%, 2%, 2% and 2%, respectively). While the fungi isolated from placenta were Aspergillus fumigatus, Candida tropicalis, Candida albicans and Mucor Spp.with incidence of (2%, 4%, 2%  and 2%, respectively).     

     In attempt to correlate the relation between various types of bacteria and fungi and their sites of positive aborted ewe foeti, the data obtained were recorded in Table (4).Out of 16 aborted goat infected with Brucella melitensis, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp. venerealis, Listeria monocytogens, Salmonella typhimurium, Salmonella dublin and Staph. aureus the organisms were present mainaly in the fourth stomach cotents with incidence of 100%  in all bacterial isolates, then marked drop in liver with an incidence of (66.7%, 66.7%, 100%, 50%, 100%, 100% and 0%, respectively), followed by spleen with an incidence of (83.3%, 33.3%, 0%, 50%, 50%, 0% and 0%, respectively).Also lung specimens were the least common seats of infection with an incidence of (33.3%, 33.3%, 0%, 0%, 50%, 0% and 100%, respectively) .

     While Out of 14 aborted ewe infected with Aspergillus fumigatus, Aspergillus niger, Candida tropicalis, Candida albicans, Mucor spp. and Rhizopus spp.The fungi were present mainaly in the fourth stomach cotents with incidence of 100%  in all fungal isolates, then marked drop in lungs with an incidence of (100%, 50%, 100%, 100%, 0% and 100%, respectively), followed by liver with an incidence of (75%, 50%, 50%, 50%, 0% and 0%, respectively).Also spleen specimens were common seats of infection with an incidence of (100%, 100%, 100%, 50%, 100% and 0%, respectively) .

     Table (5) : show the results of the antibiogram of different  isolates in which gentamicin, danofloxacin and erythromycin were the most effective anti bacterials on the Campylobacter fetus subsp.fetus, while danofloxacin, amoxicllin& clavulanic acid, erythromycin and lincomycin were the most effective anti bacterials on the Campylobacter fetus subsp venerealis, but amoxicllin& clavulanic acid, cephalocin, danofloxacin, penicillin G and chloramphenicol were the most effective anti bacterials on the Listeria monocytogens. Salmonella spp was sensitive to danofloxacin , gentamicin and chloramphenicol, but Staph. aureus isolates were sensitive to amoxicllin& clavulanic acid, cephalocin and danofloxacin , while E. coli was sensitive to amoxicllin& clavulanic acid, danofloxacin and  penicillin G.

     Table(6):summarized the results of chemical compounds were tested as antifungal agents, in which Aspergillus fumigatus and Aspergillus flavus were highly sensitive to anti fungal agents clotrimazole (canesten) and  miconazole, intermediate in sensitivity to nystatin and resistant to thibenzole.While Aspergillus niger were highly sensitive to clotrimazole (canesten) and  miconazole, but resistant to nystatin and thibenzole.  All tested strains of Candida albicans and Candida krusei completely resistant to nystatin and miconazole, but highly sensitive to clotrimazole and thibenzole. Meanwhile Candida tropicalis were highly senstitive to clotrimazole, intermediate in sensitivity to thibenzole, but resistant to nystatin andmiconazole.

      Two isolates representative of Brucella melitensis and two isolates for Aspergillus fumigatus were selected and subjected to PCR analysis. The specificity of the oligonucleotide primer was confrimed by the positive amplification of 169 and 792 bp fragments from the extracted DNA of Brucella melitensis, while 383 bp fragments from the extracted DNA of Aspergillus fumigatus (Fig.1) .

Table (1):Prevalence of bacteria and fungi isolated from vaginal discharge, placenta and aborted foeti of aborted sheep.

* Number of examined samples

Table (2): Bacteria and fungi isolated from aborted sheep foeti as regarded to its sites of isolation from the internal organs.

Table (3):Prevalence of bacteria and fungi isolated from vaginal discharge, placenta and aborted foeti of aborted goats.

* Number of examined samples

Table (4): Bacteria and fungi isolated from aborted goats foeti as regarded to its sites of isolation from the internal organs.

Table (5) :Antibiotic sensitivity  test of the different isolated strains isolated from  aborted sheep and goats using disc diffusion method.

*: Number of isolates.

S: Sensitive.

% : Percentage of sensitive isolates in relation to total isolates.

Table (6) :Antifungal sensitivity  tests of the fungi isolated from  aborted sheep and goats.

*: Number of isolates.

S: Sensitive.

% : Percentage of sensitive isolates in relation to total isolates.

                                        M              1             2            3              4

Fig.(1): Electrophoresis analysis of PCR product of amplified Brucella melitensis and Aspergillus fumigatus.

M : 100bp marker.

Lane1,2 indicate a positive amplification of Brucella melitensis at  the 169 and 792bp.

Lane 3,4 indicate positive amplification of Aspergillus fumigatus atthe 383bp.  

DISCUSSION

Abortion of sheep and goats constitute the most importante problem causes a great economic implications in terms of milk yield,  meat production and fertility of animals.

Bacteria and fungi were usually associated with abortion of sheep and goats.

In this study, the bacteriological and mycological examination of aborted foeti, vaginal discharge and placenta of (70)aborted sheep revealed that 42 samples were (60%) positive for bacteriological isolates, while 29 samples (41.4%) were positive for mycological isolates.

    As shown in Table (1), the bacteriological examination illusterated that the Brucella melitensis was the most microorganism isolated from aborted sheep with the incidence of 21.4%. They mostly isolated from aborted foeti, placenta and vaginal discharge with the incidence of(10%, 7.1%and 4.3%, respectively from sheep),followed by Campylobacter fetus subsp. fetus, Campylobacter featus subsp.venerealis with the incidence of 11.4% and 7.1%, respectively in sheep. Also Listeria monocytogens, Salmonella typhimurium, Salmonella dublin, Escherichia coli and Staph.aureus were isolatedfrom aborted foeti, placenta and vaginal discharge with an incidence 8.6%, 5.7%, 2.9%, 1.4% and 1.4%, respectively from sheep.These results agreement with Redman et al., (1963) who isolated campylobacter organisms with incidence of 14.7% from aborted ewe, Varga et al., (1990) recorded that abortion in sheep was caused in 18 flocks (78.3%) by C. fetus subsp. fetus  and in 5 flocks (21.7%) by C. fetus subsp venerealis. Also agreement with Derbala and Ghazi (2001) they isolated Brucella melitensis from aborted sheep with the incidence of 18.9%, Leyla et al., (2003) who identifed Brucella melitensis from aborted featus with an incidence of 31%.While Plagemann (1989) isolated Listeria monocytogens,  Salmonella typhimurium and Escherichia coli from aborted fetuse and placenta of aborted sheep Sargison et al., (2001) isolated E. coli from placenta of aborted sheep .

     The mycological examination of aborted sheep as shown un table (1)  illusterated that Aspergillus fumigatus was the most isolates among Aspergillus species with the incidence of 12.9%. These results agreement with Siddique et al., (1976), Cuci (1987), Vandyousefi and Zoghi (1988) and Munoz et al., (1989) they isolated Aspergillus fumigatus in a pure culture from cases of metritis and abortion in ewe. Candida albicans (8.6%), was the 2^nd isolated fungus and this is in agreement with Osman and Abou-Gabal, (1978) who isolated Aspergillus fumigatus and Candida albicans from vaginal swabs of reproductive disorder ewes, while Faried et al., (1986) reported isolation of 42.8% of infertile ewe. Aspergillus niger, Aspergillus flavus, Mucor spp. Abisidia spp. and Rhodotrula spp. were isolated with the incidence of (5.7%, 2.9%, 2.9%, 4.3%, 2.9% and 1.4%, respectively from aborted foeti, vaginal discharge and placenta of aborted sheep. This results runs parallel with Verma et al., (1999) who islated Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus  from aborted ewe and endometritis.

Specimens required for laboratory diagnosis of ovine abortions are abomasal contents, liver, spleen and lungs after fetal death. Clarifying the role of bacteria and fungi in aborted foetus may lead to more extensive light involving its problem of ovine abortion.Distribution of bacteria species and fungi species in aborted dead foeti in sheep and its incidence is represented in Table (2). It is worthy to mention that stomach of aborted ewe foetus harboured all isolated bacteria and fungi from dead aborted foeti and the stomach contents was the most common seat for isolation and considered as the specimens of laboratory choice. It was followed by liver and spleen specimens concerning bacterial isolates, but followed by lung and liver in fungual isolation.While the lungs specimens were the least organs infected by bacteria from examined aborted ewe^,s foeti, the spleen were the least infected organs by fungi.These findings coincide with the observations obtained by Redman et al., (1963) and Ardrey et al., (1972) who recovered campylobacter spp. mainly from the stomach contents of aborted ovine foetuses followed by the liver specimens, Allsup (1985) noticed that the main best sites for Campylobacter spp. were foetal liver and placenta. While Doghiem et al., (1995) reported that highest isolation of Brucella melitensis was obtained from spleen followed by liver, lungs, lymph node and kidney, Cetinkaya et al., (1999) isolated Brucella melitensis from stomach contents of aborted sheep fetuses by bacteriological isolation and by PCR.  Hunter et al., (1976) recorded isolation of salmonella typhimurium from fetal stomach contents and placenta. Low and Renton (1985) who isolated Listeria monocytogens from lung liver spleen and kidney of aborted ewe.

Isolation of  Candida albicans from stomach contents of aborted ewe foeti due to the swallowing of the amniotic fluid contaminated with the yeast bodies Smith (1967). Pier et al., (1972) isolated Aspergillus fumigatus from extrauterine organs, placenta and foetal stomach contents.Aspergillus fumigatus penetrates the placenta and infects the foetus by contamination the amniotic fluid, so stomach contents are usually a good source for isolate mycotic agents Miller (1977).

     As shown in Table (3), bacteriological and mycological examination of aborted foeti, vaginal discharge and placenta of (50)aborted goats were 32 samples (64%) positive for bacteriological isolates, while 27 samples (54%) were positive for mycological isolates. From the bacteriological examination the Brucella melitensis was the most microorganism isolated from aborted goats with the incidence of 20%. They mostly isolated from aborted foeti, placenta and vaginal discharge with the incidence of(12%,4% and 4%, respectively), followed by Campylobacter fetus subsp. fetus, Campylobacter fetus subsp. venerealis with the incidence of 10% and 6%, respectively. They mostly isolated from aborted foeti, placenta and vaginal discharge. Listeria monocytogens, Salmonella typhimurium, Salmonella dublin, Staph. aureus and Escherichia coli were isolatedfrom goat aborted foeti, placenta and vaginal discharge with an incidence 10%, 8%, 4%, 4% and 2%, respectively. These results agreement with El-Nahas (1951), reported brucellosis in goats with an incidence of (21.5%), Hamdy (1992) ) isolated Brucella melitensis from sheep and goats, Montasser (1999) tested blood serum of goat slaughtered in Cairo abattoir for Brucella was11.3% and Hosein et al., (2002) isolated Brucella melitensis from sheep and goats. Campylobacter fetus subsp. fetus isolatedin pure culture from the tissues and stomach contents of an aborted foetus of goats Anderson et al., (1983).

     Mycological examination of aborted goats shown in table(3) illusterated that Aspergillus fumigatus was the most isolates among Aspergillus species with the incidence of 14%. They mostly isolated from aborted foeti, placenta and vaginal discharge with the incidence of(8%, 2% and 1%, respectively). It was followed by Candida tropicalis (10%), Candida albicans (8%). Then  Aspergillus niger, Aspergillus flavus, Mucor spp. Rhizopus spp., Penicillium spp. and Fusarium spp. with the incidence of (6%, 2%, 6%, 4%, 2% and 2%, respectively from aborted foeti, vaginal discharge and placenta of aborted goats. This results agreement with Osman and Abou-Gabal (1978) who reported the incidence of mycotic infection of goats may reach to 50% including Aspergillus spp.and Candida spp. Agag et al., (1988) reported the isolation of Fusarium, Cladosporium and Penicillium spp. from mycotic abortion in goats.

     Bacteriological and mycological examination of the abomasal contents,liver, spleen and lungs of the goats collected from 50 aborted dead foeti as shown in Table (4), revealed that the stomach contents was the most common seat for the isolation of bacteria and fungi, followed by liver, spleen and lungs specimens for the bacterial isolation, except Brucella melitensis spleen is the second seat  followed by liver and lungs.While in mycological isolation the stomach contents followed by lungs, liver and spleen.These results are in accordance with the findings obtained by Prescott and Bruin Mosch (1981) and Andreson et al., (1983) who isolated Campylobacter spp. from stomach contents of goats. The stomach contents are usually a good source for isolation of mycotic agents (Jensen, 1990 and Jensen et al., 1994 ).     

     As shown in Table(5), most species of Campylobacter fetus subsp. fetus, and Campylobacter fetus subsp. venerealis were highly sensitive to gentamicin, danofloxacin and erythromycin but highly resistant to ampicillin, cephalocin, chloramphenicol,streptomycin and tetracycline. These findings are agreement with that obtained by Zenin (1985) and Narita et al., (1988). While amoxicllin&clavulanic acid, cephalocin, danofloxacin, penicillin G and chloramphenicol were the most effective anti bacterials on the Listeria monocytogens, these findings are agreement with Braun (2006). Salmonella spp. was sensitive to danofloxacin , gentamicin and chloramphenicol, but Staph. aureus isolates were sensitive to amoxicllin& clavulanic acid, cephalocin and danofloxacin , while E. coli was sensitive to amoxicllin& clavulanic acid danofloxacin and  penicillin G. These agreement with the results of Mishra et al., (1996).

     The results in Table (6), summarized the effects of antifungal agents on the Aspergillus fumigatus and Aspergillus flavus were sensitive to clotrimazole,  miconazole, and nystatin.While Candida albicans and Candida krusei were highly sensitive to clotrimazole and thibenzole.These results agreement with results obtained by  Rippon (1988) and Collee et al., (1996).

     The use of polymerase chain reaction (PCR), as shown in Fig. (1), revealed positive amplification of Brucellamelitensis on169bpand 792bp fragments on lane 1-2.  While lane 3-4 indicates positive amplification of 383bp fragment ofAspergillus fumigatus. These results are in agreement with the results of (Bricker and Halling 1994, Ewalt and Bricker 2000) who used AMOS PCR technique as a diagnostic assay for identification and differentiation of Brucella melitensis from other type of Brucella spp.(Abortus, Melitiensis, Ovis, Suis)  . David et al., (2005) recorded that PCR is a useful assay for detection and identifcation Aspergillusfumigatus , also is providing a good alternative to the time consuming  isolation test normally used in laboratory routine .

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الملخص العربي

البکترياوالفطريات المصاحبة للأجهاض في الأغنام والماعز في محافظة المنوفية

د/الهام ابراهيم عطوة^*               د/ فلوراج محمود راضى^**

^*قسمالبکتريولوجى^**وقسم الفطريات  بمعهد بحوث صحة الحيوان

معمل شبين الکوم

    الأجهاض يعتبر  من أهم المشاکل  التي تصيب الأغنام والماعز لما يسببه من خسائر اقتصادية کبيرة.

     وقد أجريت هذه الدراسة على (120)  عينة من الأغنام والماعز المجهضة بمحافظة المنوفية وذلک لمعرفة أهم المسببات البکتيرية والفطرية للأ جهاض فى الأغنام والماعز.

     تم  فحص الأجنة المجهضة والمخاط المهبلى والمشيمة في (70( حالة من الأغنام التى حدث بها اجهاض . وبالفحص البکتريولوجي للعينات تم عزل البروسيلا مليتينسيز والکامبيلوباکتر الجنينى تحت الجنس الجنينى والکامبيلوباکتر الجنينى تحت الجنس الفنيريالس والليستيريا مونوسيتوجين والسالمونيلا تيفيميوريم والسالمونيلا دابلين والأشيرشيا کولاى والميکروب العنقودى  بنسب (21.4% و11.4% و 7.1% و8.6% و5.7% و 2.9% و1.4% و 1.4%على الترتيب).  وبالفحص البکتريولوجي فى    (50) حالة مجهضة من الماعز تم عزل البروسيلا مليتينسيز والکامبيلوباکتر الجنينى تحت الجنس الجنينى والکامبيلوباکتر الجنينى تحتالجنس الفنيريالس والليستيريا مونوسيتوجين والسالمونيلا تيفيميوريم والسالمونيلا دابلين والميکروب العنقودى  والأشيرشيا کولاى بنسب (20% و10% و 6% و10% و8% و 4% و4% و 2%على الترتيب)   

     وبالفحص للفطريات في الأجنة المجهضة والمخاط المهبلى والمشيمة في (70( حالة من الأغنام التى حدث بها اجهاض تم عزل الأسبيروجيلس فيوميجاتس و الأسبيروجيلس نيجر و الأسبيروجيلس فلافيس والکانديدا البيکانس والکانديدا کروسي والميوکر والأبسيديا والرودوتريلا بنسب ( 12.9% و 5.7% و 2.9% و8.6% و 2.9% و 4.3% و 2.9% و 1.4% على الترتيب). ولکن بفحص الفطريات المسببة للأجهاض في (50) حالة من الماعز تم عزل الأسبيروجيلس فيوميجاتس و الأسبيروجيلس نيجر و الأسبيروجيلس فلافيس والکانديدا  تروبيکالس والکانديدا البيکانس والميوکر والريزوبس  والبنيسيليم والفيوزاريم  بنسب( 14% و 6% و 2% و10% و 8% و 6% و 4% و     2% و2% على الترتيب).

    وقد لوحظ أن محتويات المعدة للأجنة المجهضة من أهم الأماکن التي يمکن منها عزل البکتيريا والفطريات المسببة للأجهاض.

    وتم ايضا دراسة مدى حساسية العترات البکتيرية المعزولة للمضادات الحيوية فکانت معظم العترات المعزولة حساسة للدانوفلوکساسين والجنتاميسين والأموکسيسيلين مع حمض الکلافيولانک. وکانت معظم الفطريات المعزولة أکثر حساسية للکلوتريمازول (الکانيستين) و الميکونازول.

     وقد تم  أستخدام  اختبار تفاعل البلمرة المتسلسل کطريقة حديثة وسريعة لتشخيص البروسيلا مليتينسيز  الأسبيروجيلس فيوميجاتس بأستخدام البريمر المخصص لکل واحد منهم علي العترات المعزولة. وقد أثبتت النتائج سرعة ودقة أختبار تفاعل البلمرة المتسلسل فى التشخيص المعملي السريع .