Microbial Analysis of Indian Flying Fox (Pteropus giganteus) Ejecta Collected from Two Public Parks in Lahore, Pakistan

Tayiba Latif Gulraiz1, Arshad Javid1*, Syed Makhdoom Hussain2, Muhammad Shahbaz3, Irfan3 and Sharoon Daud4

1Department of Wildlife and Ecology, University of Veterinary and Animal Sciences, Lahore

2Department of Zoology, Government College University, Faisalabad

3Department of Zoology, Women University of Azad Jammu and Kashmir, Bagh

4Department of Chemistry, Forman Christian College, a Chartered University, Lahore

ABSTRACT

Microbial analysis of Indian flying fox, Pteropus giganteus ejecta roosting at Jinnah and Lalazar Gardens, Lahore was carried out from January, through December, 2011 and a total of twelve fungal and twelve bacterial genera were isolated. Four fungal (Candida, Fusarium, Penicillium and Saccharomyces) and two bacterial genera (Klebsiella and Nocardia) were isolated from bolus only, three fungal (Cryptococcus, Histoplasma and Trichophoton) and six bacterial (Acaligenes, Azotobacter, Bartonella, Nitrsomonas, Pseudomonas and Salmonella) genera were isolated from guano while five fungal (Alternaria, Aspergillus, Chrysosporium, Exophilaand Scopulariopsis) and four bacterial (Bacillus, Corynebacterium, Listeria and Streptomycete) genera were common in bolus and guano samples. Seasonal variations were recorded in occurrence of various fungal and bacterial genera. From bolus samples, two fungal Aspergillus and Fusarium and one bacterial Bacillus genera were recorded throughout the year while from guano Bacillus was the only genus with year round occurrence. Microbial analysis shows that Indian flying fox ejecta are an amalgam of beneficial and pathogenic microbes and its pH (6.7 to 7.4), high concentration of phosphorus (4.50% and 4.33%) and nitrogen (3.26% and 2.37%) favor seed germination, enhance root growth and soil fertility.

Article Information

Received 09 April 2015

Revised 26 January 2016

Accepted 08 September 2016

Available online 05 January 2017

Authors’ Contributions

TLG collected and analyzed the data, and wrote the article. SD helped in preparation of media, purification and staining of bacterial isolates. MS helped in identification of bacteria and fungi. Irfan helped in physico-chemical analysis of ejecta samples. SMH helped in interpretation of data. AJ supervised the study.

Key words

Fruit bat, Pathogen, Bolus, Guano.

* Corresponding author: arshadjavid@gmail.com

0030-9923/2017/0001-0305 $ 9.00/0

Copyright 2017 Zoological Society of Pakistan

DOI: http://dx.doi.org/10.17582/journal.pjz/2017.49.1.305.312

 

INTRODUCTION

 

Bats (Order: Chiroptera) are the only mammals capable of true flight and can cross the barriers other mammals cannot (Willson et al., 1989). They are present everywhere except Antarctica (Hutson et al., 2001) and are divided in two major groups, the Megachiroptera and the Microchiroptera. The Megachiroptera are frugivorous bats and can cover a distance of 50 km in search of food in a single night (van der Pijl, 1957). In Pakistan, fruit bats are represented by three genera and four species, the short-nosed fruit bat Cynopterus sphinx, the Indian flying fox P. giganteus, the Egyptian fruit bat Rousettus aegyptiacus and the fulvous fruit bat R. leschanaultii. However, they are least study in the country (Roberts, 1997; Shahbaz et al., 2014).

The fruit bats are important reservoirs of many pathogens, some of which have been reported to be associated with many diseases like rabies (Paez et al., 2003), European lyssa virus (Fooks et al., 2002), Hendra (Halpin et al., 2000) and Menangle (Bowden et al., 2001) in Australia, Nipah and Tioman viruses in Malaysia (Chua et al., 2002a, b) and hantaviruses in Korea (Kim et al., 1994; Chua et al., 2005). Ejecta of the fruit bats supports a great diversity of organisms including arthropods, fungi, bacteria and lichens (Ferreira and Martins, 1998) and are most common sources of pathogenic and other mycofauna distribution. The differences in composition of bats’ ejecta suggest that bats in different feeding guilds may affect ecosystem structure and dynamics (Justin and Roark, 2007).

Due to the close proximity of bats with humans and domestic animals, it is possible that they had important role in the epidemiology and zoonoses. The contact of bats with humans and domestic animals are either direct or indirect, for example through many hematophagus arthropods such as mosquitos, ticks (Pavlovsky, 1996) and cone-nosed bugs (Albuquerque and Barreto, 1968) feeds on bats, domestic animals and man. They are thought to be transferring half of the communicable diseases in man and act as a reservoir, intermediate host or vector of various pathogens (Freitas et al., 1960). The fungi related to bat excreta are mostly limited to the places where bat guano is frequently abundant (Darling, 1906). The association between bats and pathogenic fungi was first reported by Emmons (1958) who isolated Histoplasma capsulatum from soil contaminated by bat guano in Maryland. Over the next two decades, approximately 30 chiropteran species were identified as hosts for pathogenic fungi (Carvajal, 1977; Reis and Mok, 1979).

Bat guano had also reported to contain beneficial fungi and bacteria, which act as a natural fungicide to protect plants from diseases. Bacteria and fungi play important role to maintain soil health. Bacteria are necessary for plant growth on new fresh sediments. Bacteria fix atmospheric nitrogen and carbon, produce organic matter and immobilize enough nitrogen and other nutrients to initiate nitrogen cycling process in the soil (Lane and Diver, 2000). The clay is the most abundant of all the minerals in the fresh guano while others include quartz and traces of dolomite and calcite. The most abundant elements in bat guano are nitrogen and phosphorus. The total nitrogen ranges between 8–12% and P2O5 ranges between 2–7%. Other elements include calcium, magnesium, potassium, aluminum, iron and sulphur that are present in quantities lower than 5% each (Ruth et al., 2004). Differences in community structure of the microbes inhabiting guano may be due to differences in guano composition of frugivorous (P. rodricensis), sanguivorous (Desmodus rotundus), and insectivorous (Tadarida brasiliensis) bats. Desmodus guano contained more carbon than Pteropus guano. The latter contained less nitrogen, and the former contained less phosphorous than guano of the other two species. Pteropus guano had a higher C to N ratio, and Desmodus guano had higher N to P and C to P ratios than the other two species. These differences in guano composition suggest that guano from bats in different feeding guilds may affect ecosystem structure and dynamics differently (Justin and Roark, 2007).

There is a loophole of specific studies on degradation of species specific bat guano, in general the most abundant organisms in soil that contribute to organic matter decomposition are bacteria and fungi. Marinkelle and Grose (1972) documented infectious pathogenic micro-organisms isolated from bats that affected man or domestic animals. These include Salmonella spp., Spirocheaeta spp. and Leptospira spp. Numerous micro-organisms like Bartonella rochalima and Grahamella spp. etc. are also reported from the bats which are considered harmless to man and domestic animals or reported as doubtful pathogens due to the fact they are apparently not potential pathogens. There are number of medically significant fungal species isolated from the excreta of bats viz. Cryptococcus neoformans, C. laurentii, Histoplasmaca psulatum and Sporothrix schencki (Reis and Mok, 1979; Takashi et al., 2005). Keeping in view the clinical, economical, and environmental significance of fungi, bacteria and minerals found in bat ejecta the present study was designed to ascertain microbial load and mineral composition of bolus and guano of P. giganteus roosting in urban areas of Lahore.

 

MATERIALS AND METHODS

 

Study area

Present study, extending from January, 2011 to December, 2011 was conducted in two public parks i.e., Jinnah garden (35°55’ north latitude and 74°33’ east longitudes) and Lalazar garden (31°28’ north latitude and 74°14’ east longitudes) in Lahore, the second most populated city of Pakistan. The city experiences extreme summer and winter seasons, the summer season is followed by rainy and humid monsoons season. The Jinnah garden covers an area of 176 acres (0.71 km2) with Indian flying fox, P. giganteus population ranging from 3000 to 4000 individuals while at Lalazar garden which is smaller in size and stretched over an area of 04 acres (0.02 km2), the populations ranges from 800 to 1000 individuals. Both the public parks are permanent open day roosts of the Indian flying foxes (P. giganteus) and governed by Pakistan Horticulture Authority (PHA).

Sampling strategy

The ejecta (bolus and guano) of Indian flying foxes were collected by spreading a polythene sheet of 1 m × 1 m (length × width) under the roosting sites of P. giganteus. Out of total 47 plots at Jinnah garden, the P. giganteus was roosting in four plots. Four polythene sheets, one in each plot were placed at Jinnah garden while one polythene sheet was spread under the roosting canopies at Lalazar garden once a month for the whole year. Each sheet remained spread for 10 h i.e. from 2000 h Pakistan Standard Time (PST) till 6000 h PST and was removed on the subsequent day. The ejecta were randomly collected and were placed in polythene bags along with the tags indicating garden, roost number, plot number and date (Mahmood-ul-Hassan et al., 2010).

Twelve monthly samples of bolus and guano were lumped together into four seasonal samples. The February, March and April samples were named as spring sample. All the remaining monthly samples were also lumped together in the same way and designated as summer (May, June, July), autumn (August, September, October), and winter (November, December, January) samples, respectively. From each of the three monthly samples, 333.3 mg of bolus and guano was used for microbial analysis in such a way that the combined seasonal sample weighed 1 g. The pH of each seasonal sample was observed on pH meter (Mahmood-ul-Hassan et al., 2010).

Fungal analysis

Fumigated incubators, sterilized glass-wares and autoclaved apparatus were used to prevent environmental contamination. One gram of sample was transferred in 10 ml (v/w) normal saline solution to prepare five concentrations of serial dilution ranging from 10-1 up to 10-5 which were used for further identification of fungi present in the bolus and guano samples.

Sabouraud dextrose agar (16.25 g) and agar agar (5 g) media were diluted with 250 ml of distilled water, shaken, boiled, auto-claved for 45 min and poured in Petri dishes. 0.1ml of each dilution was spread on media in Petri dishes and left for incubation at room temperature for 72 h. The fungal growth was then monitored and were counted manually, petri plate with more than or equal to four fugal colonies were processed further for purification and identification while the others were neglected.

Fungal colonies were purified by picking the growth with platinum loop from each different type of colony and were placed in separate petri plates and again incubated for 72 h. The slides were then prepared from each purified colony and stained with Congo red and sealed with DPX. These permanent fixed slides were then observed under microscope (ML 5100) for identification. Macroscopic and microscopic characters of colonies were observed and genera were identified following Emmons et al. (1977).

Bacterial analysis

Seven grams of nutrient agar and 0.25 g agar agar was diluted with 250 ml of distilled water and shaken well, autoclaved for 45 min and were poured in 8 Petri plates. 0.1 ml of each dilution of the bolus and guano was spread separately on media in Petri plates and were left for incubation for 24 h at room temperature. The bacterial growth was then checked and bacterial colonies were counted on bacterial counter (Keunzahlgerat BZG 28). Those Petri plates in which bacterial colonies ranged from 30 to 300 were further processed for purification and identification while the remaining were neglected. Bacterial colonies were purified by picking the growth with platinum loop from each different type of colony and were three way streaked in separate Petri plates and again incubated for 24 h. The slides were then prepared from each purified colony, stained by gram staining method and were observed under microscope (ML 5100) for identification (Aaronson, 1970; Cruickshank et al., 1975).

Mineral composition

Each lumped seasonal sample (10 g) of both guano and bolus was incubated at room temperature in a pan for 24 h. The samples were analyzed after incubation to determine the level of different minerals present in them. The samples were weighed and dried in an oven at 105°C for 24 h and placed in separate plates. A small part of the dry matter (5 g) was ashed at 650°C for 4 h in respected crucibles (of each season) in an electric furnace. 10 ml of nitric acid was added in each of these 5 g ashed samples then the flasks were kept in water bath at 65°C for 15 min. Perchloric acid (HClO4), (5 ml) was then added to the flask and kept in water bath at 75°C for 15 min. The samples were then dried on hot plate till the fluid reached to 0.5 ml. After proper filtration, the samples were raised to 50 ml solution by repeated washing (Elaroussi et al., 1994). The mineral composition was estimated using atomic absorption spectrophotometer.

 

RESULTS AND DISCUSSION

 

During present study, a total of twelve fungal genera representing nine families were isolated from bolus and guano samples of P. giganteus. These genera included Alternaria, Aspergillus, Candida, Chrysosporium, Cryptococcus, Exophiala, Fusarium, Histoplasma, Penicillium, Saccharomyces, Scopulariopsis and Trichophyton. Nine fungal genera were isolated from bolus and eight from the guano of Indian flying fox. Genus Aspergillus (n=7) was most recorded while Histoplasma (n=1) and Trichophyton (n=1) were least recorded genera. Four of the isolated genera viz. Candida, Fusarium, Penicillium and Saccharomyces were isolated only from bolus of the Indian flying foxes whereas Cryptococcus, Histoplasma and Trichophyton were observed from isolated from guano samples only (Table I). Goveas et al. (2006) observed that P. giganteus bolus samples contain more fungi while the guano. Takashi et al. (2005) reported Candida lusitaniae and Debaryomyces hansenii from bat guano.

Seasonal variations had been observed among the nine fungal genera isolated from bolus samples of the Indian flying fox during all the four seasons viz. spring, summer, autumn and winter. Alternaria and Chrysosporium were the genera isolated only in spring, Scopulariopsis was isolated only in autumn whereas rest of the genera were isolated in two seasons as Candida and Fusariumin spring and autumn, Penicillium in spring and winter, Exopphiala in summer and autumn and Saccharomyces was isolated in summer and winter season. Aspergillus was the only genus recorded in all of the four seasons. Seelan et al. (2008) observed 23 species of bats out of which 13 (56.5%) species were found to contain 17 fungal isolates of the genus Aspergillus. Maximum numbers of fungal colonies (6.0 × 105 cfu/gm) from bolus were counted in spring season while minimum number of colonies (4.0 × 103cfu/gm) was counted in summer season (Table II). Goveas et al. (2006) documented Fusarium and Penicillium as common fungal genera in bolus and guano of Indian flying fox.

Seasonal variations were also noted regarding occurrence of fungal genera in guano samples of P. giganteus. A total of eight genera were isolated from guano during all the four seasons. Out of these, Exophiala and Histoplasma were isolated during spring season only; Scopulariopsis was isolated during summer and Trichophyton during autumn only. Chrysosporium and Cryptococcus were isolated in summer and winter season while Alternaria and Aspergillus were recorded in spring, autumn and winter. No genus was recorded in all the four seasons. Maximum number of fungal colonies (4.0 × 104cfu/gm) from guano were counted in spring while minimum number of colonies (3.0 × 104cfu/gm) were counted in autumn season (Table II). Seelan et al. (2008) documented the diversity of Aspergillus species by isolating six species of Aspergillus from 13 species of bats. The abundance and diversity of isolated fungal genera is also correlated with food sources and the roosting site of the bats. Yamamoto et al. (1995) investigated that the bat guano may mediate the exchange of pathogenic fungi just as pigeon excreta mediate the exchange of Cryptococcus neoformans, the causative agent of cryptococcosis. Apart from that, fruits consumed by these frugivore bats are also important factor in understanding the ecology of bats. Sometimes the infected fruit may contain pathogenic micro-organisms that may be present during the fruit decay process (Sepiah, 1985). So, this would be a key factor how the fungi are transmitted to bats since frugivorous bats consume fruits as their main diet.

Table I describes macroscopic and microscopic characters of all the twelve genera. Twelve bacterial genera namely Acaligens, Azotobacter, Bacillus, Bartonella, Corynebacterium, Klebsiella, Listeria, Nitrsomonas, Nocardia, Paeudomonas, Salmonella and Streptomycete were isolated from ejecta of P. giganteus. Bacillus was the only bacterial genus recorded in all the seasons from ejecta while Azotobater, Bartonella, Nitrosomonas and Sallmonella were recorded once a year.

 

Six out of ten genera isolated from only guano included Acaligens, Azotobacter, Bartonella, Nitrsomonas, Paeudomonas and Salmonella whereas two genera Klebsiella, and Nocardia were isolated from bolus samples only. Four genera Bacillus,Corynebacterium, Listeria and Streptomycete were represented in both bolus and guano samples of Indian flying fox (Table II). The bacteriological examination of bolus and guano of the Indian flying fox done by Goveas et al. (2006) revealed the presence of Alcaligenes and Pseudomonas in guano, and Bacillus, Klebsiella and Proteus in bolus. Among actinomycetes, Streptomycetes were common in guano and Micromonospora in bolus.

Mineral composition of bolus of Indian flying fox was analyzed in four seasonal samples. The pH of fruit bat bolus is near acidic to neutral ranges between 6.7 and 7.4. The most abundant elements in bolus are phosphorus and nitrogen whereas potassium is less abundant. The total nitrogen ranges between 2.28% and 4.10% in bolus which are higher than that in guano. Total phosphorus ranges between 3.50% and 5.0% and total potassium ranges between 0.6% and 0.74% (Table II).

pH of bat guano varied from 7.1 to 7.4 with nitrogen and phosphorous contents ranging from 2% to 3.30% and 3.10% to 5.20% respectively (Table II). The studies of Goveas et al. (2006) revealed higher nitrogen, phosphorus and potassium in the bolus than the guano (3.3:4.3:0.7 vs 2.6: 4.2:0.6) of the P. giganteus. Ruth et al. (2004) documented that nitrogen and phosphorus were the most abundant elements in bat guano. The total nitrogen ranges between 8–12% and P2O5 ranges between 2–7%. Other elements include calcium, magnesium, potassium, aluminum, iron and sulphur that are present in quantities lower than 5% each.

In this study, the NPK values, fungal and bacterial load in guano and bolus of the Indian flying foxes were analyzed. The result showed higher nitrogen percentage in bolus than guano whereas phosphorus and potassium percentages were higher in guano of these bats. Nitrogen in guano is known to enhance crop growth while phosphorus provokes root development, shoots, budding, multiple branches and flowering in plants. Like other bat guano studies the present study clearly indicated that ejecta of the Indian flying foxes could be significantly used with appropriate ratios with soil to increase the growth, dry matter and productivity of plant and crops.

The fungal and bacterial analyses brought forward the presence of some useful and some pathogenic genera in bolus and guano of the Indian flying fox. Among 12 fungal genera Aspergillus, Penicillium and Saccharomyces are used in drug production, food making and fermentation. The maximum species of Candida, Cryptococcus, Fusariumand Scopulariopsis are harmless and are useful soil microbes. While the genera Alternaria, Chrysosporium, Exophila, Histoplasma and Trichophyton are infectious and pathogenic either to humans, animals or plants. Among bacterial genera, Alcaligenes, Azotobacter, Nitrosomonasand Corynebacteriumhave economical and industrial significance and are also important in Nitrogen-cycle and N2 fixative bacteria in environment. Some members of Streptomycetes are pathogens while two third of them are important in medicine production and also plays important role in decaying vegetation. Nocardia species constitute major useful oral micoflora while some are pathogens. Species of Bartonella, Klebsiella, Listeria and Salmonella are infectious and pathogens. The members of Pseudomonas are important bio-control and bioremediation agents whereas some species are pathogenic.

 

CONCLUSION

 

It can be concluded from the present study that ejecta of the Pteropus giganteus are composed of beneficial and pathogenic microbes. However, it might be useful in enhancing fertility of the soil.

 

Statement of conflict of interest

Authors have declared no conflict of interest.

 

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