A PRELIMINARY SURVEY OF STAPHYLOCOCCUS AUREUS INFECTION IN GOATS WITH MASTITIS IN “NOUQRA” VALLEY OF ASWAN GOVERNORATE, SOUTH EGYPT

Assiut University web-site: www.aun.edu.eg

A PRELIMINARY SURVEY OF STAPHYLOCOCCUS AUREUS INFECTION IN

GOATS WITH MASTITIS IN “NOUQRA” VALLEY OF ASWAN

GOVERNORATE, SOUTH EGYPT

A.M.A. ZAITOUN ¹; A.E.A. ABD-EL-WAHED ²; ALSAGHER O. ALI ²

and WALAA HUSSIEN ³

¹ Infectious Diseases, Dept. Animal Medicine, Assiut Univ., Egypt

² Dept. Animal Med., South Valley Univ., Egypt

³ Vet. Directorate, Edfo, Aswan Governorate, Egypt

This article is an abstracted outline from MV-theses presented by the fourth author to Faculty of Vet. Med., Dept. Animal Med., (Infectious Diseases), South Valley University under supervision of the firs-three authors

Received: 30 December 2018;     Accepted: 23 January 2019

INTRODUCTION

Mastitis is still frequently incriminated as one of the most important threats affecting the world's dairy industry (Serrano-Rodríguez, 2017) inducing colossal damages to livestock production (Samiullah et al., 2000). There are two form of mastitis; clinical and subclinical forms. The later appears to be more prominent (Willium et al., 1987). Various pathogens are encountered as etiologic agent of mastitis. Based on the principal reservoirs of mastitogens, mastitis was into environmental and contagious mastitis (Hogan and Smith, 2012). The later appears to be more prominent than the former. Staphylococcus aureus is frequently incriminated as a serious mastitogen of milk producing goats and ewes, particularly in sublevel hygienic measures (Salaberry SR, 2015 and Serrano-Rodríguez, 2017).

Small ruminants particularly goats are more populated animals than other ruminants in El Nouqra valley. This valley is one of the oldest Egyptian valleys located in Eastern border of Nasr El Nouba and Draw centers, neighbor to “Khirt Valley” of Aswan Governorate and west to the desert of the Red Sea Governorate in South Egypt (GIS, 2013), (Fig. 1).

Corresponding author: A.M.A. ZAITOUN

E-mail address: amazaitoun@aun.edu.eg.

Present address: Infectious Diseases, Dept. Animal Med., Faculty

Assiut Univ.

Fig. 1: Centers of Aswan Governorate indicating the location of “Nouqra” Valley

The average public economic income of the working people in Nouqra valley is low (GIS, 2013) and the most people prefers goats for milk and meat productions. Moreover, Nouqra’s peoples assumed that goats have more resistance to dry and harsh environmental conditions in comparison with sheep and other large ruminates. In Nouqra valley, goat’s milk considers a preeminent food with a considerable level of nutritional value. Therefore, the aim of the current work was carried out to reveal-up the prevalence of subclinical mastitis in goats by indirect method (California Mastitis Test) in association with culturing technique focusing on Staphylococcus aureus. The isolated strains were molecularly tested to mecA gene and nuc gene of Staphylococcus aureus using PCR with species specific primers. In-vitro antibiotic sensitivity tests for the isolated Staphylococcus aureus was also done.

MATERIALS AND METHODS

A total of 148 milk samples were subsequently collected from goats of local breeds apparent normal goats with different age and parity Table (1& 3), There were four cases (2.7%) with clinical mastitis and 144 (97.3%) were apparent healthy cases. Milk samples were collected in sterile single use disposable falcon tubes with tightly fitted caps, all samples subjected to California Mastitis Test, then frozen immediately at -20^oc (Pamela, 2005).

Table 1: Age-wise distribution of goat age and their percentage.

Table 2: Average number of birth (parity).

Culturing of S.aureus

S.aureus was confirmed on baird-parker media according to (Lancette and Bannette, 2001), typical large black colonies appeared and 3-4 colonies kept in glycerol broth at -70 to-80^oc for further identification.  The coagulase test was performed by two different methods; the slide and tube coagulase test (Cookson, 1997).

Antibiotic sensitivity test

S.aureus strains which had been isolated and confirmed with coagulase test (positive samples) had been tested for it^'s susceptibility to antibiotics by disc diffusion method (Bauer et al., 1966) and (Dereesse et al., 2012), by using Muller Hinton Agar and the diameter of the zones were measured and compared (NCCLS, 2001).

Primer used in PCR assay:

Application of PCR for identification of 16srRNA, nuc gene (general primer) and mec A gene of S. aureus was carried out by using Primers as showen in the following table:

Table 3:Primers used in PCR assays.

Detection of 16s rRNA, mecA gene and nuc gene

Using Multiplex PCR method for detection of 16s rRNA of Staphylococcus genus specific, nuc gene for S.aureus species specific and mecA gene for detection of methicillin resistant S.aureus.

1. DNA extraction: A boiling procedure to the pellet at 100⁰c for 20 minutes was used to extract DNA from bacterial isolates according to Reischl     et al. (1994).

2. DNA amplification reaction: Multiplex PCR assay was performed By using total volume of 25ul reaction mix contain 5ul of template DNA, 20 pmol of each primer and 1X of PCR mix. The PCR cycles were carried out in Eppendorf AG (22331 Hamburg) thermocycler. The analysis of PCR products was carried out using 1.5% ethidium bromide stained agarose gel. This technique consists of repetitive cycles, where each cycle of PCR synthesis involves three steps: heat denaturation, annealing and extension.

3. Agarose gel electrophoresis: The agarose gel electrophoresis was performed according to Sambrook and Russell(2001). First: agarose gel is prepared and casted with concentration appropriate for the size of DNA fragments to be separated. Second: the DNA samples are loaded into the sample wells and the gel is run at a voltage and for a time period that will achieve optimal separation. Third: the gel is stained either by incorporation of ethidium bromide into the gel or electrophoresis buffer during electrophoresis or by submerging in buffer containing ethidium bromide after electrophoresis, then visualized directly upon illumination with UV light.

RESULT

All samples collected were tested by California Mastitis Test (CMT) as shown in table (4).

Table 4: CMT of collected milk samples.

By conventional culture methods on baired parker agar (table5), Two (2) samples show the characteristic colonial growth of S.aureus, 115 samples show fair growth, small black colonies and no clear zones (other staphylococci). Thirty one samples were negative.

Table 5: Frequency of the isolated S. aureus from the examined milk samples.

All staphylococci isolates were tested by slide and tube coagulase test to differentiate between coagulase positive and coagulase negative staphylococci, and found that 7 samples are positive for coagulase test and 110 samples negative for coagulase test as shown in table (6). Coagulase positive staphylococci isolates were tested for sensitivity test to 12 different antimicrobials as shown in Table (7).

All Staphylococcus spp. isolated from examined raw milk samples were subjected to PCR (Multiplex PCR) for detection of 16s rRna (for Staphylococcus genus specific), nuc gene (S.aureus species specific) and mecA gene (methicillin resistance gene), two isolates of total 7 isolates tested confirmed as S. aureus (table 8). Six (6) isolates are positive for mecA gene. Which appear as clear bands on agarose gel at a74bp compared to molecular weight marker (Figures 1, 2).

Table 6: Results of coagulase test.

Table 7: The frequency of resistance to various antimicrobials (n=7 isolates).

S. Susceptible                      I. Intermediate                R. Resistant  

Table 8: S. aureus isolates as diagnosed by PCR method.

Figure (1): PCR of nuc and mecA gene on agarosegele electrophoresis. Lane M: 100bp DNA plus ladder, Lane 1: Positive control contain 3 band (147, 228 and 279bp), Lane 2, 3:  isolates contain 3 bands of (147, 228 and 279bp) of staphylococcus aureus and mec A gene, Lane 4, 6: contain two band of (228 and 147bp) of staphylococcus but not auerus and mec A gene, Lane 5: Negative sample, Lane 7: Negative control.

Figure (2): PCR of nuc and mecA gene on agarose gel electrophoresis. Lane M: 100bp DNA plus ladder, Lane 1: Positive control contains 3 band (147, 228 and 279), Lane 2, 3: isolates contain bands of (228 and 147) of staphylococcus but not auerus and mec A gene, Lane 4: contain one band of 228 of staphylococcus but not auerus, Lane 5: Negative control.

DISCUSSION

Staphylococcus aureus—Mastitis is a widespread disease of milk producing animals including goats (Salaberry, 2015) and is associated with a significant reduction in milk yield and deteriorated milk quality. The disease results in partial or complete damage to udder tissues and decreases the productive life span of the animal (Gonzalez et al., 1980).

Currently, culturing 148 milk samples collected from mastitic and apparent normal udder of dairy goats indicated that 117(79.1%) samples gave positive result with California mastitiic test (CMT) and appear growth on baird parker agar media. Other microorganisms can produce black colonies on baired parker agar media as Enterococcus fecalis and proteus mirabilis (Baired-parker, 1992). This is agreement with (Maya et al., 2013) who found other staphylococci grow on baired parker agar as (S. schleiferi). and dis agreement with (Al-azem et al., 2013) who recorded a higher percentage of isolated strains were s. aureus (95.5%) on baired parker agar.7 isolates are staphylococcus species which gave positive result with coagulase test. The production of coagulases and thermonucleases are not unique features of S.aureus but are shared by S.intermedius and S.hyicus (El- Jakee et al., 2008).

Prevalence of subclinical mastitis of goat's raw milk obtained from Nokra Valley, Aswan Governorate, Egypt is 76.35%this result is similar to that observed by (Vasiu, 2008) as he recorded the prevalence of subclinical mastitis was 70.21%, and higher than that recorded by (Contreras et al., 2007; Leitner      et al., 2008; Bagnika et al., 2011). They found the prevalence of subclinical mastitis in goat is usually between 5 to 30%. Indiscriminate use of antibiotics has led to ineffectiveness of antibiotic treatment (Ali et al., 2010).

Resistance of staphylococci to methicillin and all β-lactam antibiotics is associated with the low affinity of a penicillin-binding protein, PBP2a, which is not present in susceptible staphylococci. Pierre et al. (1990); Unal et al., 1992; Chamber, (1997). This protein is encoded by the mecA gene. Mastsuhashi (1986), is the corner-stone responsible for producing MRSA phenomenon (Ubkata et al., 1989; Berger-Bachi 1997).

It is concluded that, Staphylococcus aureus—subclinical mastitis is seriousness problem in goat’s population in the area of study. The misuse of antimicrobial agents leading to the development of resistant isolates which may be transmitted to the human beings causing somber troubles. Amplification of DNA by PCR is a rapid and sensitive method for the detection of specific DNA sequences.

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