Isolation of aerobic bacteria from ticks infested sheep in Iraq



Isolation of aerobic bacteria from ticks infested sheep in Iraq

E-mail: zenadaboodi@yahoo.com

Peer review under responsibility of Hainan Medical University.

Waleed Ibrahem Jalil1, Mohammad Mushgil Zenad2*1Department of Veterinary Internal and Preventive Medicine, College of Veterinary Medicine, University of Diyala, Diyala, Iraq

2Department of Veterinary Internal and Preventive Medicine, College of Veterinary Medicine, University of Baghdad,

Baghdad, Iraq

ARTICLE INFO

Article history:

Received 27 Jul 2015

Received in revised form 10 Aug, 2nd revised form 24 Aug 2015

Accepted 28 Sep 2015

Available online 10 Nov 2015

Keywords:

Ticks

Aerobic bacteria

Sheep

Iraq

ABSTRACT

Objective: To highlight the presence of aerobic bacteria in live ticks infested sheep, in Diyala Governorate, Iraq.

Methods: One hundred and thirty adult alive ticks were picked up from sheep which were reared in different farms in Diyala Governorate, Iraq, during the period from November 2012 to May 2013. Ticks were classified in the Natural History Museum in Baghdad. They were dissected aseptically for extraction of the salivary gland and midgut. The removed tissue from each organ was inoculated in buffer peptone water (1%) and incubated for 2 h at 37°C, to maintain weak and/or injured bacterial cells, then transmitted to nutrient broth incubated at 37°C for 18 h. Culturing was done on three solid bacteriological media (nutrient, blood and McConkey agars), and then incubated at 37°C for 24 h. Bacterial identification was performed by using multiple biochemical tests and API-20 strips. Data were analyzed by using Statistical Analysis System version 9.1, 2010. Chi-square test was used for comparison at significant level of P≤0.05.

Results: Two species of ticks were identified [Rhipicephalus (Boophilus) annulatus and Hyalomma turanicum]. High bacterial isolation rate was observed (483 isolates). A significant high isolation rate was recorded from Rhipicephalus annulatus (63.14%). Six bacterial species were identified [Escherichia coli (28.36%), Pseudomonas aeruginosa (18.01%), Bacillus cereus (14.69%), Staphylococcus aureus (13.66%), Citrobacter freundii (13.04%), and Enterobacter species (12.21%)]. Also the high bacterial isolation rates were recorded in the temperate months (November, March and April); these coincided with high reproductive performance of ticks.

Conclusions: The high isolation rate of aerobic pathogens from ticks might reflect the active contribution of this arthropod in environmental contamination and increase the probability of transmitting bacterial pathogens to their hosts.

1. Introduction

Ticks are the most harmful arthropod and infest different animal species and man [1]. They cause high economic losses among animal production [2]. The undesirable direct effects of tick's parasitism are anemia and skin lesions, resulting in damage of wool and leather, which ultimately interfere with their industries. Moreover, paralysis of different host species may occur due to tick's salivary toxins [3]. In addition, their serious role is transmitting many various pathogens via mechanical or biological ways[4]. Some of these pathogens are life-threatening to animals [5], whereas others have a great risk to human health [6,7]. Furthermore, it was believed that more than 100000 diseased conditions in human being in the world belonged to the tick borne infection [8]. Few studies had been done to clarify the role of ticks in transmission of bacterial organisms. Several researches showed that many bacterial species were isolated from salivary gland and mid-gut of various tick's species [9]. The incidence of tick borne diseases increased according to variation in weather of different environments, particularly when suitable conditions are provided for growth and reproduction of ticks [10]. It wasreported an increment in infection rates of Borrelia burgdorferi and Anaplasma phagocytophilum, occurred in consequence to seasonal variations [11]. The current article is a preliminary study in Iraq, aimed to shed light on the extent of bacterial pathogens lurking in salivary glands and mid-guts of hard ticks infested sheep in Diyala Governorate, Iraq.

2. Materials and methods

2.1. Ticks

One hundred and thirty live adult ticks were collected from Awassi and Hamdani sheep, during the period from November 2012 to May 2013. The approval of this study had been taken from the Council of Veterinary Medicine College/University of Baghdad and the work was performed complied with the current Iraqi laws. Ticks were gently picked up by hands using wetted cotton piece with 70% ethyl alcohol[12]. Ticks were classified in the Natural History Museum in Baghdad[13]. Dissection of ticks was achieved under high aseptic condition (bacteriological hood) [14]. A sterile phosphate buffer solution was added to dissected tissues of ticks to avoid dryness.

2.2. Bacterial culture

The removed salivary gland and mid-gut were inoculated in buffer peptone water and incubated for 2 h at 37°C, in order to maintain and reanimate the weak microorganisms and repair the injured bacterial cells, then one full bacteriological loop was transmitted into nutrient broth and incubated at 37°C for 18 h. Culturing was done on three solid media (nutrient, MacConkey and blood agars) and they were incubated at 37°C [15]. The isolated bacteria were classified by using multiple biochemical tests and API-20 test strips (BioMerieux, Inc.).

2.3. Statistical analysis

Data were analyzed by using Statistical Analysis System (version 9.1, 2010) and Chi-square test was used for comparison at significant level of P≤0.05.

3. Results

Two species of ticks were identified [Rhipicephalus (Boophilus) annulatus (R. annulatus) and Hyalomma turanicum (H. turanicum)]. Four hundred and eighty-three isolates were recorded from all collected ticks (130). The significant (P≤0.05) higher total isolation rate was recorded from R. annulatus (63.14%) whereas from H. turanicum, the total isolation rate was 36.85%. Moreover, the significant (P≤0.05) increases of bacterial isolation from R. annulatus were observed during all months of the study. Furthermore, a significant increase of Escherichia coli (E. coli) (137) isolates was recorded from both ticks species, followed by Pseudomonas aeruginosa (P. aeruginosa) (87), Bacillus cereus (B. cereus) (71), Staphylococcus aureus (S. aureus) (66), Citrobacter freundii (63) and Enterobacter spp. (59) (Figure 1).

Figure 1. Isolates of bacterial species from ticks.A: E. coli; B: P. aeruginosa; C: B. cereus; D: S. aureus; E: Citrobacter spp.; F: Enterobacter spp.

The isolation rate increased significantly in April (28.36%) and decreased to a minimum value in January (5.28%) and the elevation of bacterial isolation rates seemed to coincide with temperate months of November, March and April (Figure 2).

Figure 2. Isolation rates of bacterial species from ticks according to months.Max: The maximum value rates of temperature (°C) during months; Min: The minimum rates of temperature (°C) during months.

The insignificant higher total isolation rates of bacterial species were recorded from viscera of both tick species: R. annulatus (161) and H. turanicum (94), whereas those from salivary gland (144, 84 in both species of ticks respectively) were recorded significant higher total isolation rates, with exception of S. aureus (Table 1).

4. Discussion

Ticks are important blood sucking ectoparasite causing anemia, skin lesion and they produce tick paralysis in hosts [3]. In addition, they play a serious role in transmission of many pathogens to various host species and some are life-threatening [16,17]. Few literatures were reported on the role of ticks in transmission of bacterial pathogens in animals. In fact, ticks are highly prevalent in Iraq, which hinder the development of farm animal production. This was a preliminary study aimed to highlight the extent of bacterial species resided within tissues of ticks infested sheep. The high isolation rate of bacterial organisms from both tick species (483 isolates), referred to the contribution of this arthropod in contamination of the environment, rather than transmission of these bacteria to their hosts. The different species of bacteria protect themselves from external harmful influences of the environment inside the body of tick and perpetuate their life in nature [18,19]. In the same instance, the ticks play a role in an evolutionary process of bacteria and might lead to create new bacterial strains or change their virulence [20,21]. The total isolation rate of bacterial organisms were significantly (P≤0.05) higher from R. annulatus (63.1%) than from H. turanicum (36.8%). This pointed to that tick species is a more serious reservoir for many pathogenic bacteria. It was also reported that certain tick species harbor pathogens while others do not [22]. The E. coli was an abundant (28.3%) bacteria, isolated from both tick species, which was in agreement with other researches [18]. Such a redundancy of E. coli isolates might be due to wide prevalence of this organism in the nature and the extensive fecal contamination among sheep farms might probably assist in increasing ticks infection (or harboring such pathogens). According to the increased isolation rate of E. coli bacteria reflected the unhygienic sanitation as well as the bad management, these ultimately increased the pollution of environment with E. coli. Six bacterial species were recorded in both tick species; some researches showed similar bacterial isolates whereas others showed different isolates according to various regions and times [18,19,23]. However, other authors found many various species of bacteria in ticks related to certain ecological conditions [23,24]. Other different microbes including many bacterial species were isolated not only from adult ticks but also from their eggs, larvae and nymphs [24,25]. Moreover, it was reported that female ticks had less diverse microbiomes than males and nymph [26]. Indeed, these stages of life increase the probability of bacterial transmission and furthermore, the harboring of many different bacterial species by ticks reflects the variations in ecology and environments. A marked increment of bacterial isolation was observed in the temperate months: March (mean of minimum and maximum temperatures was 9.88 and 24.39°C respectively) and April (14.02 and 30.67°C respectively), which was coinciding with the reproductive activity of ticks. As the environmental temperature and rain fall [10,27], were highly suitable for optimum tick reproduction, ticks come out of their slumber and looked for suitable hosts. This gives an explanation for the presence of various bacterial species inside ticks in different areas. Ticks may take infection from environment or from contaminated wool or skin of the hosts. Simultaneously, this presence in the period is favorable for growth and multiplication of bacteria. The non-significant higher isolation rate from mid-gut (52.8%) of ticks, compared with salivary glands (47.2%), was recorded. Moreover, all isolated bacterial species showed higher isolation rates from viscera of ticks (except S. aureus), than that from salivary glands and on the contrary, others found higher infection rate in the salivary glands [9]. The residence of ticks in salivary gland increases the risk to their hosts and it was reported that tick's salivary secretions have roles in modulation of host defense mechanisms and pathogen transmission[28].

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

This work was performed as M.Sc. project in Baghdad University by Waleed Ibrahem Jalil under my supervision. It was supported by the College of Veterinary Medicine/University of Baghdad.

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Original article http://dx.doi.org/10.1016/j.apjtb.2015.10.002

*Corresponding author:Mohammad Mushgil Zenad, PhD, Department of Veterinary Internal and Preventive Medicine, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq.