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In the Name of ALLAH,

Most Gracious, Most Merciful

Volume (7) – NO.(1)

January 2014 – Rabi I 1435H

Scientific Publications & translation

Deposit No.: 1429/2022

EDITORIAL BOARD

Editor-in-Chief:

Prof. Hassan M. Mousa

Editorial Board:

Prof Fahad A. Al-Sobayil

Prof. Mohamed Abd El-Sattar El-Meleigi

Prof. Ansary E. Moftah

Prof. Moustafa M.Zeitoun

Advisory Board:

Prof. Abdulrahman I. Al-Humaid, Saudi Arabia

Prof. Hani M. Gohar, Egypt

Prof. Abdrab Al-Rasoul Omran, Saudi Arabia

Prof. Heungshik S. Lee, Korea

Prof. Hamzah M. AboTarboush, Saudi Arabia

Prof. Hassan A. Melouk, USA

Prof. Steven D. Lukefahr, USA

Prof. Ibrahim M. Al-Shahwan, Saudi Arabia

Prof. Mohamed M. Youssef, Egypt

Prof. Jean Boyazoglu, France

Prof. Abdulmageed M. Kamara, Egypt

Prof. Maher H. Khalil, Saudi Arabia

Prof. William E. Artz, USA

Prof. Ghanem M. Al-Ghamdi, Saudi Arabia

Journal of Agricultural and Veterinary Sciences,

Qassim University, Vol. 7, No. 1, pp. 1-99 Eng, pp 1-24 Arabic (January 2014/Rabi I 1435H)

Contents Page

English Section

Veterinary Medicine Survival of Brucella melitensis Rev-1 in experimentally contaminated and

Refrigerated camel and goat milk Khaled B. Alharbi ............................................................................................................................. 3

Plant Production and Protection A Checklist to the poisonous plants of Qassim Region, Saudi Arabia

Gamal E.B. El Ghazali and Hassan M. Mousa ............................................................................. 21

Field evaluation of some insecticides against the pink bollworm,

Pectinophoragossypiella and the spiny bollworm, Eariasinsulana Laila R. Elgohary ........................................................................................................................... 35

Food Science and Human Nutrition Nutritional Status of Pregnant Women in Relation to Anthropometric Measurements

of Mothers and their newborns Mahmud R.M.; I.S. Ashoush and H.M. Harb and Soha H. Mahmud........................................ 45

Physico-Chemical, Rheological and Sensory Properties of Date Phoenix dactylifera

Var. Shamia Sheets Abd El-Hady A. El-Sayed, Youssef, Khaled M., Shatta, Adel A. and El-Samahy, Salah K. .... 59

Food Processing Wastes: Characteristics, Treatments and Utilization Review Abdalla S.M. Ammar..................................................................................................................... 71

Use of Microbiological Parameters to Verify the Application of Hazard Analysis

Critical Control Point (HACCP) during Ice Cream Production Rizk. A. Awad, Hassan, Z. M. R., M. M. Abd El-Razik and Ahmed, T. S ................................. 85

Arabic Section A Survey of Plant-Parasitic Nematode Genera in Al-Qassim Governorates Reyiad

Al-Khabraa , Oyuon Al-Gawaa , Arrass , Al-Nabhaniyah S.A. Al-Rahiayani ......................................................................................................................... 24

V

Veterinary Medicine

Journal of Agricultural and Veterinary Sciences,

Qassim University, Vol. 7, No. 1, pp. 1-99 Eng, pp 1-24 Arabic (January 2014/Rabi I 1435H)

Journal of Agricultural and Veterinary Sciences

Qassim University, Vol. 7, No. 1, pp. 3-17 (January 2014/Rabi I 1435H)

3

Survival of Brucella melitensis Rev-1 in experimentally contaminated and

Refrigerated camel and goat milk

Khaled B. Alharbi

Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim University,

P. O. Box 6622 Buraydah 51452, Qassim, Saudi Arabia

e-mail: [email protected]

(Received 15/10/2013, accepted 5/12/2013)

ABSTRACT This research work aimed to examine the survival of Brucella melitensis Rev-1 in refrigerated camel and goat milk. Fresh camel and goat milk was experimentally contaminated with

different concentrations of colony forming units per ml (CFU/ml) of Brucella melitensis Rev-1 and

refrigerated for different periods of time. Brucella melitensis Rev-1 was detected in the refrigerated contaminated milk by isolation in culture medium and by PCR. Brucella melitensis Rev-1 was isolated

from camel milk samples refrigerated for up to 8 hours. Camel milk samples refrigerated for 24 hours

were negative for bacterial isolation. Brucella melitensis Rev-1 was isolated from camel milk samples containing 65 (CFU/ml) while samples containing less than that were negative for culture. The minimum

content of the bacterium to be detected by PCR in contaminated camel milk was 65 CFU/ml. The higher

the bacterial content of milk the better is the isolation results and whole milk was superior to cream for isolation. Brucella melitensis Rev-1 was isolated from goat milk refrigerated for up to 72 hrs. Isolation

was positive for samples containing as low as 3 CFU/ml refrigerated for 4 hrs. Brucella melitensis Rev-1

was isolated from contaminated goat milk samples containing 30 to 300 CFU/ml refrigerated for 24 and 48 hrs, respectively. Samples containing higher bacterial concentrations were positive for isolation after

72 hrs of refrigeration. PCR was positive for Brucella melitensis Rev-1-contaminated goat milk

containing as low as 3 CFU/ml. These results show that both isolation and PCR are sensitive for the detection of milk contamination with Brucella melitensis Rev-1, and that Brucella melitensis Rev-1 will

not survive in camel milk for more than 8 hrs. the difference found here may be attributed to the nature of

milk itself.

Keyword: milk, goat, camel, Brucella melitensis Rev-1, PCR

mailto:[email protected]

Khaled B. Alharbi 4

INTRODUCTION

Brucellosis is a zoonotic disease globally affecting mainly domestic animals causing genitourinary infections leading to abortion (Brooks et al., 2012). The main pathogenic species are B. abortus, responsible for bovine brucellosis, and B. melitensis, the main aetiologic agent of ovine and caprine brucellosis (Ali et al., 2013).

Brucella microorganisms localize in the supra-mammary lymph nodes and mammary glands of the infected animals (Bamaiyi et at., 2012) and thus may continue to be secreted in milk throughout life (Hamdy and Amin, 2002). Milk is the foremost source of Brucella infection in humans and its testing by bacteriological culture is made difficult by the low sensitivity of analytical tests as well as by the periodicity of organism shedding in milk (Ning et al., 2013). Therefore, there is a need for the development of more accurate and sensitive tests capable of detecting low number of Brucella organisms in infected milk (Gwida et al., 2011).

Dairy products prepared from unpasteurized milk such as soft cheeses, yoghurts, and ice creams may contain high concentration of the bacteria and consumption of these is an important cause of human brucellosis (Earhart et al., 2009).

Camel brucellosis is an important zoonotic disease in the middle east and Saudi Arabia, a country with 10 million sheep, 6 million goats, 300,000 cattle and 400,000 camels (WHO, 1999; Mantur and Amarnath, 2008). The prevalence of human brucellosis in Saudi Arabia has reached an alarmingly high level in recent years, which is believed to be due to a large extent to the consumption of raw camel milk, a widely spread habit among the population of this country. Based on different serological tests, camel brucellosis incidence among camel herds in Riyadh region was found to be from 1.89 to 4%. All camels that tested positive to brucellosis were clinically normal at the time of sampling and none had previous history of disease that could be ascribed to brucellosis although some herds from which the samples were obtained reported previous history of abortion and/or mastitis (Qurashi, 2006).

Camels are very susceptible to brucellosis and under extensive farming conditions high prevalence rates of the disease have been reported in this species. Camels can be infected by B. abortus and B. melitensis when they are pastured together with infected sheep, goats and cattle (Musa, 1995; Musa et al., 2008; Gwida et al., 2012). Caprine brucellosis is an endemic in most countries at the Mediterranean basin, Middle East and Central Asia (Seleem et al., 2010 and Samadi et al., 2010)

Although camel brucellosis has been reported by serologic tests, isolation of Brucella microorganisms from camel milk has been difficult. Some researchers have failed to isolate Brucella organisms from the milk of seropositive camels after milk was transported to the laboratories and processed for culture within 1 – 2 days (Jawad, 1984; Al Khalaf and El Khaladi, 1989; Agab, 1993; Obeid et al., 1996). Isolation of Brucella sp. was possible from some but not all of the milk samples obtained from seropositive camels (Radwan et al., 1995). However, the isolation of Brucella sp. from internal organs of infected camels (particularly lymph glands, testes and vagina) was found to be relatively easy (Agab et al., 1996; Ramadan et al., 1998).

Survival of Brucella melitensis Rev-1... 5

This study was planned to investigate survival of Brucella in camel and goat milk subjected to refrigeration.

MATERIALS AND METHODS

Milk

Fresh camel and goat milk were obtained from brucella-free, non-vaccinated animals. The teat orifice was swabbed with 70% ethyl alcohol and milk was collected in a narrow opening sterile tube held in oblique position (Hawari and Hassawi, 2008). Milk was transported while cold to the laboratory to be processed within 30 minutes.

Bacteria

Brucella melitensis vaccinal strain Rev. 1 (Lio-vac rev-1, laboratories syva s. a., Spain) was used in the study. The vaccine was obtained from Qassim Veterinary Directorate, Saudi Ministry of Agriculture.

Experimental inoculation: The vaccine was reconstituted in the supplied diluent and a loopfull was inoculated onto tryptic soy agar plates. The inoculated

plates were incubated at 37C and examined after 3 days for colonial growth. Suspected colonies were identified using modified Ziehl-Neelsen staining method and polymerase chain reaction. Growing colonies were used for experimental inoculation of fresh camel and goat milk and for DNA extraction.

Inoculation of milk with B. melitensis Rev-1 strain

A bacterial suspension was prepared by transferring B. melitensis Rev-1 colonies into 5 ml of sterile physiological saline to make heavy suspension. After thorough mixing of the bacterial suspension, 1 ml was transferred to 9 ml camel milk to make 1/10 dilution. One millilitre of the 1/10 bacterial dilution in milk was transferred to 9 ml camel milk from which one millilitre was transferred to 9 ml milk and so on to make bacterial 10 fold serial dilution in camel milk up to 10

-10 dilution

from the original bacterial broth suspension. Goat milk was similarly inoculated with B. melitensis Rev-1 strain. Each milk dilution was distributed into 19 tubes, 0.5 ml

each and kept in the refrigerator at 4C. Aliquotes from each dilution were frozen for the polymerase reaction assays. Meanwhile, bacterial colony forming units (CFU)/ml stock saline suspension were counted using the plate count method.

Isolation of B. melitensis from milk

Milk samples were centrifuged at 500 xg for 20 minutes. Loopfuls of both cream and sediment were streaked onto surface of selective brucella agar (brucella agar and supplement “Sigma, UK” with 5% fresh horse serum). The inoculated plates were incubated at 37°C and examined daily for up to 10 days. Bacterial smears were stained with Gram and modified Ziehl-Neelsen methods and examined under oil emersion lens. Colonies with colonial and microscopical characters of brucella (glistening and acid fast) were confirmed with PCR (Ilhan et al., 2008).

Khaled B. Alharbi 6

DNA extraction from milk samples

Extraction of DNA from milk samples was performed according to the classical method of Lusk et al., (2013). Frozen milk samples were thawed at room temperature and 400 μl of lysis solution and 10 μl of proteinase K (10 mg/ml) were added to 400 μl of the thawed milk sample. The contents were incubated at 50°C for 30 min with shaking. Thereafter, 400 μl of tris-saturated phenol (liquid phenol containing 0.1% 8-hydroxyquinoline, saturated and stabilized with 10 mM Tris-HCl (pH 8) and 0.2 % of 2-mercaptoethanol) were added, mixed thoroughly and centrifuged at 8,000 x g for 5 min. The aqueous layer was transferred to a fresh tube and an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added, mixed thoroughly and centrifuged as described above. The upper layer was again transferred to a fresh tube and an equal volume of 7.5 M ammonium acetate was added and mixed thoroughly. The contents were kept on ice for 5 min, centrifuged at 8,000 x g for 5 min and the aqueous content was transferred to a fresh tube. Two volumes of 95% ethanol were added, mixed and the tubes were stored at –20 °C for 12 h. DNA was recovered by the final centrifugation as described above, the pellets were rinsed with 1 ml of 70% ethanol, dried and resuspended in 50 μl TE buffer (Ilhan et al., 2008).

DNA extraction from B. melitensis Rev-1

B. melitensis Rev-1 strain was grown on tryptic soy agar plates for 5 days. A loopfull of the growing colonies was carefully suspended in 400 μl of lysis buffer. After mixing the bacteria with lysis buffer DNA was extracted as described with the milk samples (Leal-Klevezas et al., 1995; Bardenstein et al., 2002).

Polymerase Chain Reaction

Two Brucella melitensis-specific oligonucleotides were used as primers for the PCR assays. Both primers target the Brucella omp2 gene (Bardenstein et al., 2002). Primer sequences are 5'-TGGAGGTCAGAAATGAAC-3' forward and 5'-GAGTGCGAAACGAGCGC-3' reverse. The oligonucleotides were synthesized and supplied by Eurofins MWG Operon (Ebersberg, Germany). PCR amplification was performed in 25 µl volumes according to the method of Bardenstein et al. (2002) and Helmy et al. (2007) with modifications. Illustra

TM puReTaq Ready-To-Go PCR

Beads (GE Healthcare, UK Limited, Amersham Place, Buckinghashire, UK). To each bead, 5 µl of extracted DNA, 15 picomole of each primer and 13 µl sterile deionized water were mixed. The mixture was overlaid with a drop of mineral oil. Tubes were held in a thermocycler (GeneAmp PCR System 9600, Perkin-Elmer Corporation, Norwalk, CT, USA). Amplification program was set up for the reaction

as following: one step at 95C for 3 minutes, 35 cycles of denaturation, annealing

and extension steps at 95C for 30 seconds, 50C for 1 minute and 72C for

1minute, respectively. A final extension step was done at 72C for 7 minutes. At the end of the program, tubes were taken out of the thermocycler to run electrophoresis on 1% agarose in 1X TAE buffer containing ethidium brobide (0.5 mg/ml). DNA bands were visualized on a UV transilluminator and the gel was photographed using a digital camera (Canon PC1210, Canon Inc., Japan).


RESULTS

Isolation of Brucella melitensis Rev-1 from camel milk inoculated with the

bacteria

Isolation of Brucella melitensis Rev-1 from inoculated milk samples containing different bacterial cell concentrations was carried out. Inoculated milk tubes were refrigerated for 3 days (since it gave negative results of isolation after 24 hours) and bacterial isolation was done at different time intervals. From table (1), it is noticed that Brucella melitensis Rev-1 was successfully isolated from milk samples until 8 hours from the inoculation time. After 24 hours refrigeration, Brucella melitensis Rev-1 could not be isolated from any of the inoculated tubes either of high or low bacterial concentration. Tubes with bacterial concentrations of 6.5 x 10

5 to 65 CFU/ml milk resulted in positive isolation while tubes with less than

65 CFU/ml resulted in negative isolation. It is also important to notice that tubes with higher bacterial content resulted in better isolation results and sediment was superior to cream layer concerning the positive isolation. Milk tubes with 65 bacterial CFU/ml showed positive isolation from sediment after 4 hours and from cream after 8 hours but were negative during the first 2 hours and at and after 24 hours. Tubes with 650 bacterial CFU/ ml were negative for isolation after 30 minutes and 2 hours while positive at 1, 4 and 8 hours post-inoculation. Tubes with bacterial CFU from 6.5 x 10

3 to 6.5 x 10

5 resulted in positive isolation at 30 minutes,

1 hour, 2 hours, 4 hours and 8 hours.

Table (1). Isolation of Brucella melitensis Rev-1 from refrigerated camel milk tubes containing different bacterial CFU/ml

Duration from

inoculation time

Bacterial cells/ 1 ml milk

6.5 x 105 6.5 x 104

6.5 x 103

6.5 x 102 6.5 x

10 6.5 0.65

30 minutes + (1) + (1) + (1) _ _ _ _

1 hour + (1&2) +(1 &2) +(1 &2) +(1 &2) _ _ _

2 hours +(1) +(1) +(1) _ _ _ _

4 hours +(1) +(1) +(1) +(1) +(1) _ _

8 hours + (1) + (1) + (1) + (1) + (2) _ _

24 hours _ _ _ _ _ _ _

48 hours _ _ _ _ _ _ _

(+): Brucella melitensis Rev-1 isolated

(-): No brucella isolated

(1): Isolation from milk sediment

(2): Isolation from cream layer

Khaled B. Alharbi 8

Isolation of Brucella melitensis Rev-1 from goat milk inoculated with the bacteria

Isolation trials of Brucella melitensis Rev-1 from milk containing different bacterial cell concentrations was carried out. Inoculated milk tubes were refrigerated for 5 days and bacterial isolation was done at different time intervals. Isolation results are depicted in table (2). From the table, it is noticed that Brucella melitensis Rev-1 was successfully isolated from milk samples until 72 hours from the inoculation time with 3 x 10

7 to 3 x 10

3 CFU/ml bacterial content. Tubes with bacterial concentrations

of 3 x 107 to 3 CFU/ml milk resulted in positive isolation until 4 hours from the

inoculation time. Tubes with 3 CFU/ml resulted in positive isolation until 4 hours only while tubes 30 CFU/ml and 300 CFU/ml were positive for brucella isolation up to 24 and 48 hours post-inoculation refrigeration, respectively. Sediment was also superior to cream layer concerning the positive isolation.

Table (2). Isolation of Brucella melitensis Rev-1 from refrigerated goat milk tubes containing different bacterial CFU/ml

Duration from

inoculation time

Bacterial cells/ 1 ml milk

3 x 107 3 x 106 3 x 105 3 x 104 3 x 103 3 x 102 3 x 10 3

2 hours + (1 & 2) +(1 & 2) +(1) +(1) +(1) +(1) +(1) +(1)

4 hours +(1 & 2) +(1 & 2) +(1 & 2) +(1 & 2) +(1& 2) +(1& 2) +(1&2) +(1&2)

24 hours +(1 & 2) +(1 & 2) +(1 & 2) +(1 & 2) +(1& 2) +(1) +(1) _

48 hours +(1 & 2) +(1 & 2) +(1 & 2) +(1 & 2) +(1& 2) +(2) +(2) _

72 hours +(2) +(2) +(2) +(2) +(2) _ _ _

(+): Brucella melitensis Rev-1 isolated (-): No brucella isolated (1): Isolation from milk sediment (2): Isolation from cream layer

PCR on DNA extracted from camel and goat milk containing different numbers of brucella CFU

Polymerase chain reaction assays, using Brucella melitensis Rev-1-specific primers, were performed on DNA extracted from camel milk samples containing different concentrations of Brucella melitensis Rev-1 bacterial cells (from 6.5 x 10

5

to 0.65 CFU/ml. Bacterial DNA was successfully amplified and the expected size (282 bp) specific for Brucella melitensis Rev-1 was visualized on UV transilluminator after agarose gel electrophoresis. Product was obtained with DNA extracted from milk containing 6.5 x 10

5 down to 65 x 10

3 CFU/ml. Below 65 x 10

3

Brucella melitensis CFU/ ml camel milk, the PCR product band was fuzzy and not that clear (figure 1). Polymerase chain reaction assays were also performed on DNA extracted from goat milk samples containing different concentrations of Brucella melitensis Rev-1 bacterial cells from 3 x 10

7 to 3 CFU/ml. Bacterial DNA was

successfully amplified and the expected size (282 bp) specific for Brucella


melitensis Rev-1 was visualized on UV transilluminator after agarose gel electrophoresis. Product was obtained with DNA extracted from milk containing as little as 3 Brucella melitensis CFU/ ml milk (figure 2).

Figure (1). Products of PCR assay using Brucella melitensis Rev-1 specific primers on DNA extracted from camel milk containing Brucella melitensis Rev-1. The 282 bp product is specific for Brucella melitensis Rev-1 DNA. M: 500 bp DNA size marker, lane 1: DNA from milk containing 6.5 x 105 brucella CFU/ml, lane 2: DNA from milk containing 6.5 x 104 brucella CFU/ml, lane 3: DNA from milk containing 6.5 x 103 brucella CFU, lanes 4 to 7 DNA from milk containing less than 6.5 x 103 CFU/ml, lane 8 has no loaded samples and lane9 is negative control (No template DNA)

Figure (2). Products of PCR assay using Brucella melitensis Rev-1 specific primers on DNA

extracted from goat milk containing Brucella melitensis Rev-1. The 282 bp product is specific for Brucella melitensis Rev-1 DNA. M: 500 bp DNA size marker, lane 1: DNA from milk containing 3 x 107 brucella CFU/ml, lane 2: DNA from milk containing 3 x 106 brucella CFU/ml , lane 3: DNA from milk containing 3 x 105 brucella CFU, lanes 4 DNA from milk containing 3 x 104, lane 5: DNA from milk containing 3 x 103 brucella CFU/ml, lane 6: DNA from milk containing 3 x 102 brucella CFU/ml, lane 7: DNA from milk containing 30 brucella CFU/ml, lane 8: DNA from milk containing 3 brucella CFU/ml

Khaled B. Alharbi 10

DISCUSSION

B. melitensis colonies were recovered from inoculated-refrigerated camel milk when

cultivation was done at 30 minutes, 1 hour, 2 hours, 4 hours and 8 hours post-

inoculation. Sensitivity of the isolation was found to be undulating in relation to the

post-inoculation refrigeration period. This was indicated by successful brucella

isolation at 30 minutes from milk tubes containing 6.5 x105 to 6.5 x10

3 bacterial

CFU/ ml but not from tubes with lower that content. At one hour cultivation,

brucella colonies were obtained from milk tubes containing 6.5 x102 brucella CFU/

ml milk. Thus, the sensitivity was increased. At 2 hours, the sensitivity was

decreased one log to be similar to the 30 minutes cultivation. At 4 and 8 hours,

sensitivity of isolation of brucella from camel milk was so increased that bacterial

colonies were obtained from milk tubes containing as little as 65 brucella CFU/ ml

milk which was likely the lowest brucella count that could be detected by isolation

from camel milk. At 24 and 48 hours post-inoculation, no brucella could be isolated

from any inoculated tubes. This was also met with cultivation at 48.

The increased sensitivity of isolation at 1, 4 and 8 hours than at 30 minutes

post-inoculation can be explained by the possible engulfment of bacterial cells by

phagocytes that may be present in milk as Brucella is an intracellular bacteria.

Another explanation is the interference of antibacterial agents that might be present

in the milk and act during the first hour such as lysozymes.

The available literature concerning this and suggestion the current

investigation. Although camel brucellosis has been reported by serologic tests that

one of the most challenges is the isolation of Brucella microorganisms from camel

milk. Some researchers have failed to isolate Brucella organisms from the

milk of seropositive camels after milk was transported to the laboratories and

processed for culture within 1 – 2 days (Agab, 1993; Obeid et al., 1996) and

others reported the isolation of Brucella spp. from some but not all of the milk

samples obtained from seropositive camels (Radwan et al., 1995). However, the

isolation of Brucella spp. from internal organs (particularly lymph glands, testes and

vagina) was found to be relatively easy (Agab et al., 1996; Ramadan et al., 1998).

When applied on DNA extracted from milk samples containing B. melitensis

Rev-1 microorganisms, the PCR assay succeeded in the detection of the bacteria DNA

and the expected product was obtained. However, sensitivity of the PCR assay was not

too much superior to the isolation. PCR wasn't able to detect brucella DNA in camel

milk containing less than 6.5 x 103 CFU/ ml milk which was successfully obtained

with isolation after 4 and 8 hours post inoculation. This finding does not likely agree

with the known higher PCR sensitivity than traditional isolation with most species of

bacteria and most clinical samples. Camel milk contains substances that may interfere

with the PCR and minimize its sensitivity. However, the PCR in this case is surely

superior to cultivation when time factor is considered as Brucella colonies do not

usually appear on plates before 3 days of incubation and even after growth the

growing colonies require further identification that requires more time. It was


recommended that conventional PCR could be used in the developing countries

because of the lower costs and the high amount of Brucella DNA in the samples of

infected camel, in contrast to other animal species. They added that real time PCR was

most sensitive and specific when compared to conventional PCR (Gwida et al., 2013).

Concerning goat milk, sensitivity of both cultivation and PCR for detection

of B. melitensis was better than that obtained with camel milk. At 2 and 4 hour post-

inoculation refrigeration, cultivation from goat milk resulted in isolation of B.

melitensis from tubes with bacterial counts of 3x107 CFU/ ml to tubes with 3 CFU/

ml milk. At 24 and 48 hours post-inoculation cultivation B. melitensis was isolated

from goat milk tubes containing at 3x107 and 3x10 CFU/ ml milk. Whereas, at 72

hours post-inoculation cultivation B. melitensis was isolated from goat milk tubes

containing at 3x107 and 3x10

3 CFU/ ml milk.

When PCR was applied to detect B. melitensis Rev-1 in goat milk inoculated

with different bacterial counts, the assay was found to be so sensitive that the

specific 282 bp band was clearly obtained from DNA extracted from milk tubes of

both high and low bacterial number. DNA extracted from goat milk containing as

little as 3 CFU of B. melitensis / ml resulted in a clear PCR product as detected by

agarose gel electrophoresis. Data confirmed previously by ALGaradi et al., (2011)

who reported that PCR was the most sensitive technique as 95.5 % of B. melitensis

in goat. PCR also has the added advantage of being able to simultaneously detect B.

abortus and B. melitensis and differentiate them with high sensitivity and specificity

(Mirnejad et al., 2012)

In a similar investigation, PCR assay amplified Brucella-DNA from 29

bovine milk samples, 10 from sheep, 13 from goats and one from a camel.

Meanwhile, the direct culture method detected Brucella organisms from 24 samples

of cows' milk, 12 from sheep, 10 from goats and failed to detect any Brucella

organisms from camels' milk. PCR detected up to 100 colony forming units (CFU)

of B. abortus per millilitre of milk in 100% of diluted milk samples, and 1000 CFU

of B. melitensis from 70% of milk samples (Hamdy and Amin, 2002).

The differences, found in this study, between survival of B. melitensis in

camel milk and goat milk can be attributed to the component of milk itself (Elagamy

et al., 1992). It has been noted that despite the lack of refrigeration, camel’s milk

remains unspoiled for several days: this may be due to the antibacterial activity of

certain minor proteins contained in camel’s milk (Elagamy et al., 1996).

Milk of mammals is protected to different extents against microbial

contaminations by natural inhibitory systems, including the lactoperoxidase /thiocyanate

/hydrogen peroxide system (LPS), lactoferrins, lysozyme, immunoglobulins and free

fatty acids (Elagamy et al. 1992; Kappeler et al. 1999). The concentration and the

activity of each of these antimicrobial systems/substances depend on the animal species

and on the stage of lactation. Camel’s milk is reported to have a stronger inhibitory

system than that of cow’s milk (Elagamy 1992). In particular; the levels of lysozyme and


lactoferrins are reported to be two and three times higher than those of cow’s milk,

respectively (De Valdez et al. 1988; Kappeler 1999).

In a previous study on the microbiological quality of Moroccan camel’s milk,

it was observed that coliform organisms were not always present in the samples

analysed, despite the high total aerobic counts in the same samples. Such results

suggest that coliforms are more sensitive to the inhibitory systems of camel’s milk

than other groups of microorganisms (Benkerroum et al. 2003).

It was also demonstrated that camel milk had a bacteriostatic effect against E.

coli and bacteriostatic to L. monocytogenes, while the colostrum was bactericidal,

suggesting that different antimicrobial systems occurring in camel’s milk may be

responsible for the inhibition of each of the pathogens tested (Benkerroum et al.,

2004). The protective effect against E. coli of raw camel’s milk was found to be

clearly higher than that of heated milk, which suggested that the heat treatment

process may destroy, at least partially, some of the inhibitory systems present in the

milk. The lysozyme is sensitive to heat and the LPS was shown to be completely

inactivated when heated at 80°C for 40 s or at 76°C for 1 min (Marks et al.2001;

Benkerroum et al., 2004). Such kinds of antibacterial substances in camel milk

could lead to disappearance of B. melitensis from the milk tested in this study within

24 hours after introducing bacteria into the milk.

ACKNOWLEDGEMENTS

Thanks to Prof. Mahmoud Hashad, Department of Veterinary Medicine, College of

Agriculture and Veterinary Medicine, Qassim University for his professional

assistance.

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جامعة القصيم –كلية الزراعة والطب البيطري –قسم الطب البيطري

م(1/53/3152م ؛ قبل للنشر 51/51/3152يف للنشر )قدم

يف حليب اإلبل و املاعز املربد. 5استهدفت هذه الدراسة بقاء بروسيال ميلتتزس ريف

حيث مت تلويث حليب اإلبل و املاعز الطازج واملربد جتريبيا مع تركيزات خمتلفة من وحدات تشكل

و لفرتات زمنية خمتلفة. ومن ثم مت الكشف عن 5املستعمرات لكل مل من بروسيال ميلتتزس ريف

عزل من املستنبتات و استخدام تفاعل البلمرة يف احلليب امللوث بواسطة ال 5الربوسيال ميلتتزس ريف

من عينات حليب اإلبل املربدة اجيابيًا ملدة 5. وقد كان نتائج عزل بروسيال ميلتتزس ريفPCRاملتسلسل

ساعة. 32ساعات. بينما كانت نتائج العزل سالبه يف عينات حليب اإلبل املربدة وامللوثة بعد 8تصل إىل

وحدة مستعمره، يف حني كانت سالبه لليت حتتوي على أقل 51ينات اليت حتتوي على وكان العزل اجيابيا للع

من ذلك. وكان احلد األدنى من احملتوى البكتريي الذي ميكن الكشف عنه بواسطة تفاعل البلمرة املتسلسل

PCR يب كانت وحدة مستعمره لكل مل. و كلما زاد احملتوى البكتريي يف احلل 51يف حليب اإلبل امللوث هو

نتائج العزل أفضل وكلما كان احلليب كامل الدسم كانت نتائج العزل أفضل. يف املقابل كانت نتائج عزل

ساعة. وكان العزل 23من عينات حليب املاعز املربدة اجيابيه ملدة تصل حتى 5بروسيال ميلتتزس ريف

ساعات. 2مل من احلليب املربد حتى وحدة مستعمره لكل 2اجيابيًا للعينات اليت حتتوي على ما يصل إىل

21211بنجاح من عينات حليب املاعز امللوثة واليت حيتوي على 5كما مت عزل بروسيال ميلتتزس ريف

ساعة على التوالي. بينما العينات اليت حتتوي على تركيزات أعلى 28و 32وحدة مستعمره لكل مل ملدة


يد. وكان تفاعل البلمرة التسلسلي إجيابيًا للربوسيال ميلتتزس ساعة من الترب 23كانت إجيابية ا لعزل حتى

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النتائج أن كال من العزل و تفاعل البلمرة التسلسلي تستطيع الكشف عن تلوث احلليب بربوسيال ميلتتزس

8ال تستطيع البقاء على قيد احلياة يف حليب اإلبل ألكثر من 5كما أن بروسيال ميلتتزس ريف، 5ريف

ساعات. وميكن أن يعزى هذا إىل طبيعة احلليب نفسه.

.، تفاعل البلمرة املتسلسل5احلليب ،املاعز ، اإلبل، بروسيال ميلتتزس ريف


Plant Production and

Protection



21

A Checklist to the poisonous plants of Qassim Region, Saudi Arabia

Gamal E.B. El Ghazali (1) and Hassan M. Mousa (2)

(1) Al Rass College of Science and Arts, (2) College of Agriculture and

Veterinary Medicine, Qassim University, Saudi Arabia

E-mail address [email protected]

(Received 10/10/2012, accepted 5/1/2013)

Abstract. This study aims to define the poisonous plants that grow in Qassim Region, and affect human and animal health. A total number of (42) species belonging to (39) genera and (23) families were

recorded. These plants were encountered through questionnaires conducted with herbalists and owners of

Perfumery shops "Attarren" deployed in the region as well as shepherds, nomads and citizens of expertise and experience in the field of poisonous plants in the region. Members of the family Poaceae (5

species) were reported as the most dominant toxic flowering plant family, followed by members of the

families Chenopodiaceae and Boraginaceae (4 species each). These poisonous plants accounts for about 10 % of the total flora of the Region. For each species cited, botanical and vernacular names, family,

toxic part and the toxic chemical principles are documented

Key words: native plants, toxic plants, toxic principles, vernacular names

Gamal E.B. El Ghazali and Hassan M. Mousa 22

Introduction

Qassim Region is a leading agricultural region in Saudi Arabia, and occupies almost

the Northern Centre of the Arabian Peninsula. It is situated between latitude 24° 25'

– 27° 15' North and longitude 41° 30' – 45° 41' East, and is bounded by Hail

Region in the North and Northwest, Al Madina Region in the West, and Riyadh

Region in the South and East (map. 1).

A poison is a substance that, in substantial amount, produces adverse health

effects by causing injury, illness, or death, when ingested, inhaled, absorbed or

injected into the body of the organism. Poisons may be environmental pollutants,

household products, pesticides, industrial chemicals, food additives, drugs or natural

toxins.

A toxin, a more specific term, is any of various poisonous substances that are

specific products of metabolic activities of the living organisms. (Merriam- Webster

Dictionary). The terms "poisonous" and "toxic", although are quite unique, a

distinction between them is not always observed and are regarded as synonyms.

Plants have developed a wide array of defense mechanisms to avoid, tolerate or

defend themselves against natural enemies such as predators, parasites and

competitors. There are several categories of defenses that have been evolved in plants,

including structural (mechanical), and chemical defenses (Patrick et al. 1997).

Structural (or mechanical) defenses include prickles, spines, awns, thorns, or

barbs or even sharp edges of certain parts, which can penetrate the skin and cause

injury, itching and irritation of the victim.

Chemical defenses are chemical compounds produced by plants as secondary

metabolites or as by-products from the essential functions of the plant, and are

stored in vacuoles in the plant cells.

A vast array of biologically active chemical principles are found in the plant

Kingdom. Many of these compounds have positive (medicinal or therapeutic) and

few have negative (toxic) physiological effects which are responsible for the toxicity

of most poisonous plants (Foster & Caras 1994). These physiological effects lead to

enormous economic losses in the region where they grow (Aganga et al. 2011,

Torrell et al. 2000, Panter et al. 2002).

Poisonous plants are inevitable part of our habitat and are of widespread

distribution as wild species in streets, parks and in pastures, or in doors as

ornamental plants (Heussner et al. 2009, Lynn et al. 2005). Many of these plants are

also ethnobotanically used for treatment of various diseases in Humans and animals

(Asgarpanah & Ramezanloo 2012), and just destroying them because being

poisonous, represent a significant economic loss. Instead of trying to ovoid or get rid

of them, becoming educated about poisonous plants is the best solution to establish a

sustainable ecosystem and sustainable utilization of natural resources.

A Checklist to the poisonous plants... 23

The present study aims to document the poisonous plants of Qassim Region

and to cite the chemical toxic principles they possess in an attempt to be aware of

their harmful physiological effects and the possible use of their therapeutic

properties.

Material and Methods

The material of the present study was identified from two main sources of

information. The first source of information was obtained from questionnairing

herbalists and owners of perfumery shops "Atarreen" deployed in the region,

herders, nomads and citizens in close connection with wildlife and aware of the

benefits and harms of plants to humans and animal health. References and published

scientific articles represent the second category of sources of the present study.

Regular field trips in different seasons were conducted in Qassim Region, in

the period from May 2011 to July 2012, to collect plants reputed (from

questionnaires), to possess poisonous physiological effects in an attempt to confirm

their identity and to document their scientific names. The plant specimens collected

were identified according to Chaudhary (2001, 2000, 1419, 1989), Chaudhary & Al

Jowaid (1999), Collenette (1985), Migahid (1990, 1989, 1988, 1978). The plants

identified were confirmed by comparison with authenticated specimens in regional

herbaria. Voucher specimens were pressed, dried using blotting papers at room

temperature and mounted in herbarium sheets, and deposited at the Faculty of

Science & Arts at Al Rass, Qassim University.

For each species, an intensive literature survey was conducted to cite

published principle toxic principles with toxic biological effects.

Results

A total number of (42) species were reputed to exhibit toxicity to human beings and

animals in Qassim Region. These species belong to (39) genera and (23)

angiospermic families. Members of the family Poaceae (5 species) were reported as

the most dominant toxic flowering plant family in the Region, followed by members

of the family Chenopodiaceae and Boraginaceae (4 species each). A list of these

plants, their families, vernacular names, toxic parts and toxic principles are

summarized in table 1.


Figure (1). Poisonous plants of Qassim Region. (A) Anagallis arvensis L. (B) Cleome ambyocarpa

Barr. & Murb. (C) Salsola imbricata Forssk. (D) Convolvulus arvensis L.

Figure (2). Poisonous plants of Qassim Region. (A) Lepidium sativum L. (B) Malva parviflora L. (C)

Melilotus indica (L.) All. (D) Pergularia tomentosa L. .


Figure (3). Poisonous plants of Qassim Region. (A) Rhiza stricta Decne. (B) Solanum nigrum L. (C)

Suaeda vermiculata Forssk. (D) Withania somnifera (L.) Dunal.

Discussion and conclusions

A total number of (42) poisonous plants were encountered in Qassim Region

belonging to (39) genera and (23) angiospermic families. According to Al- Turki

(1997), the flora of Qassim Region was represented by 450 species belonging to

257genera and 62 families. In the light of the latter study, the present checklist

showed that about 10 % of the species, 15 % of the genera and 37 % of the families

of the plants of the Region are poisonous.

Most of the poisonous plants documented for the Region are also reported

elsewhere. At the regional level, (17) species out of the (42) species reported for

Qassim Region were also reported in Hail Region (Sharawy & Al Shammari 2009).

Some poisonous plants e.g. Calotropis procers, Ricinus communis, Solanum nigrum,

are of wide distribution in tropical and subtropical regions and are well reputed to

possess toxic effects (El Ghazali et al. 2008, Duke & Ayensu 1985, Watt & Breyer-

Brandwijk 1962, Oliver-Rever 1986). Other species encountered in the Region are

well known at the generic level to exhibit toxicity worldwide e.g. Euphorbia spp.,

Heliotropium spp., Chrozophora spp., Oxalis spp. (Frohne & Pfander 2004, Cooper

et al. 2003, Kingsburg 1964). In the absence of studies on the endemic species of the

Region, and the fact that the toxic chemical principles of most of the plants of the


Region are already studied, it is difficult to decide (if not impossible) the number

claimed in the Region to be poisonous and not poisonous elsewhere.

Acknowledgements

The authors gratefully acknowledge the generous financial support provided

by the Deanship of Scientific Research, Qassim University.

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كلية الزراعة والطب البيطري، جامعة القصيم، اململكة العربية السعودية. (2)كلية العلوم واآلداب بالرس و (1)

(م5/1/2112قبل للنشر يف ، م11/11/2112) قدم للنشر

وتىثرر على ، منطقىة القصىيم يف الى تنمىو السىامة النباتىا التعرف على تهدف هذه الدراسة إىل

هىذه مت حصىر فصيلة نباتية. (22و) جنسا (23) نوعا تنتمي إىل (22مت توريق عدد ) .صحة اإلنسان واحليوان

البىدو وكىذل حمىال العطىارا املنتشىرا يف املنطقىة أصىحاب مى ال أجريى من خالل االستبانا النباتا

أظهىر الدراسىة أن يف املنطقىة. النباتا السامة يف جمال اخلربا والتجارب من ذو واملواطنني والرعاا الرحل

اليصىىا ل أعضىىا ، تليهىىا يف املنطقىىة للنباتىىا السىىامةهىىم األك ىىر ً ىىيال األنىىوا ( 5) أفىىراد اليصىىيلة النجيليىىة

النباتا الى جممو من ٪11 ً ل حوالي السامة النباتا هذه لكل منهما(. األنوا 2)الرمرامية والرباجنية

العامية، باإلضافة إىل والعلمية األمسا ال مت التعرف عليها، مت توريق األنوا نو من لكل يف املنطقة. تنمو

الكيميا ية اليعالة. والعناصر السامةاليصيلة النباتية، واألجزا

النباتا الطبيعية، النباتا السامة، املادا السامة اليعالة، األمسا العامية.



35

Field evaluation of some insecticides against the pink bollworm,

Pectinophoragossypiella and the spiny bollworm, Eariasinsulana

Laila R. Elgohary

Faculty of Agriculture, Mansoura University, Mansoura, Egypt

Received 12/6/2013,accepted 5/9/2013) )

ABSTRACT. The present study was conducted to evaluate the efficacy of six commercial insecticides

namely Lampada super, Pstoxkz, Engeo, Cygron, Chlorosan and Feroban against pink bollworm,

Pectinophoragossypiella (saunders) and spiny bollworm,Eariasinsulana (Boisd.)at Aga district, Dakahlia Governorate, Egypt during 2010 and 2011 cotton growing seasons. The obtained results indicated that, all

tested insecticides exhibited great reduction in pink bollworm and spiny bollworm infestation. The

treatments could be arranged descendingly according to the general reduction averageof two seasonsas follows; Pstoxkz (94.2%), Cygron (89.3%), Engeo (88.1%), Feroban (87.5%), Chlorosan (86.7%) and

Lampada super (77.5%) against pink bollworm; and were Cygron (97.6%), Pstoxkz (95.1%), Chlorosan

(90.9%), Engeo (87.4%),Lampada super (86.1%) and Feroban (85.8%) against spiny bollworm.


INTRODUCTION

Cotton, the world major fiber crop is perhaps unique in the broad nature of the insect

attack to which it is subjected and the control of cotton insect pests remains an

unabated challenge (Johnstone, 2006).In Egypt, about half million cotton feddan

were cultivated in 2006 cotton growing season that represent about 6% of all

cultivated area. Pests are such serious threat to cotton production and the cost of

cotton pests control is about $12.5 million(Younis et al 2007).Pink bollworm,

Pectinophoragossypiella(Saund.) and the spiny bollworm, Eariasinsulana(Boisd.)

are the most serious cotton pests in Egypt (Hussein et al. 2002). They caused the

greatest yield losses from nearly one million hectares cultivated annually in the

world (Haque 1991; El-Naggar 1998). The spiny bollworm larvae damage buds

early in the growingseason and squares, and bolls later in the season.The reduction

in cotton yield was mostly related to the late season infestation with two species and

the economic yields are almost impossible to achieve without their chemical control

(Younis et al 2007).

The objective of the present study is evaluate the effect of the field

recommended rate of six commercial insecticides ; Lampada super, Pstoxkz, Engeo,

Cygron, Chlorosan and Ferobanagainst two cotton bollworms pink bollworm,

P.gossypiella (Saund.) and spiny bollworm, E.insulana (Boisd.)

MATERIALS AND METHODS

Tested insecticides:

Six commercial insecticides were evaluated in this study. The tread name, active

ingredient and recommended field rate of tested insecticide are shown in Table (1).

Table (1). Active ingredient and recommended field rate of tested insecticide.

Tread name Active ingredient recommended

field rate

Lampada super 10% WP Lambda-cyhalothrin 50g/100 L

Pstoxkz 15%EC Alpha-cypermethrin 165ml/fed.

Engeo 24.7% SC Thiamethoxam 14.1% + Lambda-cyhalothrin 10.6% 160 ml/fed.

Cygron 10% EC Flufenoxuron 3% + Alpha-cypermethrin7% 250 ml/fed.

Chlorosan 29% EC Chloropyrifos 24% + Cypermethrin 5% 750 ml/fed.

Feroban 50% EC Chloropyrifos 47.5% + Lufenuron 2.5% 1 L/fed.

Field experiment:

Experiments were conducted during 2010 and 2011 cotton growing seasons

at Aga district, Dakahlia Governorate, Egypt. Giza 86 cotton variety was used. The

A Checklist to the poisonous plants of Qassim Region, Saudi Arabia 37

design of experiment was conducted in a Randomized Block Design. The area

divided into seven treatmentscontained control area. Each treatment contains four

replicates. The area of each replicate was 175 sq. meter. All the normal cultural

operations were carried out in the experimental plots.Each of the tested insecticides

was applied three times at two weeks intervals.FirstSpraying was applied on 21th

and

26th

of July during both cotton seasons 2010 and 2011. A knapsack sprayer provided

with one nozzle delivering (200 liters water / feddan). Samples of 100 green bolls

per treatment (25 bolls for each replicate) were taken at random and dissected.

Percents of infestation were estimated immediately before the first spray and then

every week throughout the period of experiment. The reduction percentages were

calculated according to Henderson and Tilton (1955).

RESULT AND DISCUSSION

Results in Table (2), showed the efficiency of six commercial insecticides against

the pink bollworm larvae, P.gossypiella (Saund.) during three sprays in

2010,2011seasons. The obtained results indicated that, based on the reduction

average, percentage in infestation of pink bollworm after first spray in 2010cotton

season was ranged between 60% to 100 %. Engeo and Pstoxkz were the most

effective compounds as they caused 100% reduction, followed by Lampada super

and Cygron causing 90% and 80% reduction, respectively while Chlorosan and

Feroban were the least effective which reached to 65% and 60%, respectively. In

2011cotton season the percentage of reduction in pink bollworm larval population

was lesser than 2010cotton season after the first spray and could be arranged

descendingly as follows Feroban 86.7% followed by Lampada super and Cygron

80%, Pstoxkz 75%, Engeo70% and Chlorosan was the least effective which reached

to55%. According to general reduction average of two seasons, it was clear that

Pstoxkz induced the highest effect 94.2% reduction in larval population while

Cygron suppressed the number of pink bollworm larvae by 89.3% followed by

Engeo 88.1%, Feroban 87.5% and Chlorosan 86.7%. The least effective was

recorded by Lampada super 77.5%.

Data present in Table(3), showed the effects of the same commercial

insecticides against the spiny bollworm larvae, E.insulana (Boisd.) were recorded

during three sprays in 2010,2011 cotton seasons. The obtained results indicated that,

based on the average of two seasons reduction, after first spray, Pstoxkz and Cygron

were the most effective compounds as they caused 100 %.In 2010 season we could

be arranged reduction average descendingly as follows, Chlorosan, Cygron and

Pstoxkz 100 %, Feroban 91.7%, Engeo 83.3% and Lampada super 75%. While in

2011 season Chlorosan, Lampada super andEngeo wear recorded nearly the same

value 83.8%, 83.3% and 83.3% reduction, respectively. Feroban was the least

effective recording 66.7% reduction. According to general reduction average of two

seasons , the data was indicated thatCygron induced the highest effect 97.6%

reduction in larvae population followed by Pstoxkz95.1% and Chlorosan 90.9%


while Engeo, Lampada super and Feroban were recorder 87.4%, 86.1 and

85.8%ofreduction, respectively .

Insecticide efficacy depends on the initial activityof the active ingredient on

the target pest and its residual activity (persistence), which are both influenced by

environmental parameters such as temperature, sunlight, or rainfall (Mulrooney and

Elmore, 2000). The efficiency of the pyrethroids depend upon the active chemical

groups in each pyrethroid, nature and ratio of optical and geometric isomers which

allow a certain degree of effectiveness, the physical properties which determine the

degree of penetration and volatility which can increase or decrease the efficiency

(Abo-Sholoaet al. 2000). In this respect, the efficacy of the tested commercial

insecticides on the pink and spiny bollworms in the field could be demonstrated

according to the differences in their chemical structure and formulations. Perusal of

these results clearly exhibited that, all tested compounds were gave a good reduction

in the pink bollworm and spiny bollworm infestation in two seasons at the initial and

residual activity.

These results agree with those obtained by Khattaket al. (2004), khan et al.

(2007) and Balakrishmanet al. (2009), who indicated that Karate 2.5% EC (lambda-

cyhalothrin), Sherpa 5% EC (cypermethrin) and bifenthrin 10% EC at their

recommended rates were more effective in reducing the incidence of bollworms.

Zidanet al. (2012) mentioned that, the tested pyrethroid insecticides (α-cypermethrin

and Lambda-cyhalothrin) were more efficient in controlling the bollworms larval

population than carabamate and organophosphrus compounds. El-Basyoui (2003)

found that synthetic pyrethroids were the most efficient compounds compared with

organophosphrus and carbamate insecticides of the larvae of bolloworms. Also

Younis et al. (2007) reported that, the synthetic pyrethroid, Lambda-cyhalothrin and

deltamethrin exhibited the greatest reduction in bollworms infestation compared

with the organophosphrus pesticide treatment (chlorpyrifos and profenofos).

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