Prevalence of intestinal parasites with molecular detection and identification of Giardia duodenalis in fecal samples of mammals, birds and zookeepers at Beni-Suef Zoo, Egypt

Abstract

The current study aimed to investigate the prevalence of intestinal parasites from various species of mammals and birds housed in a zoological garden in Beni-Suef province, Egypt. A total of 77 fecal samples were collected from various primates (16), carnivores (7) and herbivores (54). Meanwhile, 123 fecal samples were collected from two Ostrichs (Struthio camelus), five Numida meleagris (Numida meleagris), twoIndian Peafowls (Pavo cristatus), two Emu (Dromaius novaehollandiae) 101 Pigeons (Columba livia domestica) and 11 Swan Goose (Anser sygnoides). In addition, seven stool samples from zookeepers who had been in close contact with animals and birds were examined. Salt flotation and formol ether sedimentation techniques were applied for parasitological examination. Positive samples of Giardia cysts were preserved in alcohol and kept at 4 °C until DNA extraction. Parasitological findings revealed that 48.05% of zoo animals were infected with intestinal parasites; 11.69% were positive with helminths and 27.27% with protozoa, however 9.09% had mixed infection. It was found that 75%, 57.14% and 38.89% of primates, carnivores and herbivores respectively were infected with intestinal parasites. In Primates the most prevalent parasites were Giardia spp. (43.75%) then Entamoeba histolytica/dispar (12.5%), Entamoeba coli (12.5%) and Trichuris spp. (6.25%). For carnivores, Ancylostomatidae had the highest prevalence (42.86%) followed by Spirometra spp. (14.29%). Meanwhile, Eimeria spp. (20.37%) was the most prevalent parasite in herbivores, followed by Blantidium coli (7.41%) and Tricuris spp. (7.41%), then Toxocara spp. (3.70%). Furthermore, the prevalence of infection in zoo birds was 21.95%. The identified parasites were Eimeria spp., Giardia spp., Capillaria spp., Ascaridia spp., Isospora spp. and Sublura brumpti. Stool examination of zookeepers revealed the presence of G. doudenalis and E. histolytica/ dispar cysts. The gdh gene of G. duodenalis was successfully amplified from fecal samples of zoo mammalsand zookeepers. In conclusion, the application of preventive and control measures against the propagation of infectious intestinal parasites is essential to prevent the spread of these parasites among zoo animals or to humans.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12639-020-01341-2.

Introduction

Now, zoological gardens stay to preserve animal endangered species through captive breeding and reintroduction programs (Mitchell and Tully 2009; Maesano et al. 2014). Zoo mammals and birds (wild and domestics) have an essential role as hosts and reservoirs of many zoonotic parasites (Mirzapour et al. 2018). When these wild animals and birds preserved in captivity in zoological gardens, the problem of parasitic infections can increase (Muoria et al. 2005). The gastrointestinal parasitic infections are one of the serious health problems causing diseases and even mortality (Thawait et al. 2014). Some factors control the propagation of parasites in mammals and birds kept in zoological gardens as the type of husbandry practices, disease prophylaxis and treatment administered (Lim et al. 2008).

Unfortunately, little information on parasitic diseases of zoo mammals and birds is a major limiting factor in zoological gardens (Rahman et al. 2014). Many of these parasites are zoonotic pathogens (Purcell and Philipp 2005).Human and non-human primates having the same ecosystems may sometimes lead to sporadic outbreaks of zoonosis (Lee et al. 2010). As well, Unhygienic handling of workers with animals enlarges the probability of zoonotic transmission (Adejinmi and Ayinmode 2008). Examination of intestinal parasites in zoo animals and birds has veterinary importance to restrain the spread of intestinal parasitic diseases to human and domestic animals (Mir et al. 2016; Mirzapour et al. 2018). Undoubtedly, a permanent program of gastrointestinal parasite monitoring and means of control measures established on accurate diagnosis, effective treatment and appropriate prophylaxis would help in reversing the condition of ill health in zoo mammals (Rahman et al. 2014). Studies on parasitic diseases of wildlife are still insufficient, so the present investigation attempts to determine the prevalence of enteric parasites in zoo mammals and birds in Beni-Suef province, Egypt. Besides, the zoonotic potential of these pathogens in zookeepers was estimated.

Giardia duodenalis (syn Giardia intestinalis) is a major protozoan parasite that infects a large variety of animals and humans (Thompson 2000; Hunter and Thompson 2005). Molecular detection and genotyping of G. duodenalis in animals and humans is currently underway (Read et al. 2004; Fallah et al. 2008). The application of molecular diagnostic skills in the genotypic determination of G. duodenalis has led to increased identification of parasites that infect humans and animals and the importance of animals in the spread of human giardiasis (Xiao and Fayer 2008). Detection of G. duodenalis genotypes has carried out established on the characterization of the small subunit ribosomal RNA (SSU-rRNA), β-giardin (bg), glutamate dehydrogenase (gdh) and triose phosphate isomerise (tpi) genes (Plutzer et al. 2010). The gdh gene is useful for the genotypic detection of G. duodenalis parasites from mammals (Hazrati Tappeh et al. 2014).

Therefore, The aim of this investigation is necessary to establish a perfect perception of the gastrointestinal parasite fauna of these mammals and birds to prevent the spread of zoonotic parasites among mammals in the zoo or to humans besides the detection of the gdh gene of G. duodenalis in fecal samples of wild mammals and zookeepers in the zoo garden, Beni-Suef province.

Material and methods

Study site and period

The current study was conducted during the period from November 2018 to June 2019 at zoo garden in Beni-Suef province (coordinates: 29°04′N 31°05′E), Egypt. Beni Suef province has a hot desert climate (BWh) in Köppen-Geiger classification, as does all of Egypt. It has very hot summers and warm winters with cool nights. Beni-Suef zoo is one of the zoos in Egypt and is affiliated with the Central Administration of Zoos. It was established in 1996 and opened in 1997; it reaches 4 feddan and contains many species of zoo mammals and birds.

Animals and birds husbandry (housing)

This study covered the various families of mammalsand birds housed in Beni-Suef zoo. A total of 77 zoo mammals of 12 species and 123 birds of six species were investigated. Animals were classified into non-human primates, carnivores and herbivores animals. Non-human primates included 10 Grivet monkeys (Chlorocebus aethiops) housed in four boxes, four Hamadryas baboons (Papio hamadryas) held in a large caged area and two Brown capuchin (Cebus apella) kept in a single box. Carnivores were five African lions (Panther Leo) and two Hyena (Crocuta crocuta) each animal kept in a separate box. Meanwhile, herbivores animals involved one Lamma (Lama glama), one Shetland pony, 34 Mountain goats (Capra aergagrus), 12 Barbary sheep (Ammotragus lervia), two European deers and three Hippo (Hippopotamus amphibius) beside one Nile lechwe (Kobus megaceros). Each herbivore species was housed in a separate yard. Concerning birds were classified into sixspecies including two Ostrichs (Struthio camelus), five Numida meleagris,, twoIndian Peafowls (Pavo cristatus), two Emu (Dromaius novaehollandiae), 101 Pigeons (Columba livia domestica) and 11 Swan Goose (Anser cygnoides), each bird species were housed in a separate yard. Data of the examined mammalsand birds were obtained from labels on the cages and boxes of each species.

Samples and sampling process

Fecal samples collection

A total of 77 and 123 fecal samples were collected from 12 species of mammals and six species of birds respectively at zoo Beni-Suef. Animals' samples involved 16 samples from three species of non-human primates (10 Grivet monkeys (Chlorocebus aethiops), four Hamadryas baboons (Papio hamadryas) and two Brown capuchin (Cebus apella), seven samples from two species of carnivores (five African lions (Panther Leo) and two Hyena (Crocuta crocuta). and 54 samplles from seven species of herbivores(one Lamma (Lama glama), one Shetland pony, 34 Mountain goats (Capra aergagrus), 12 Barbary sheep (Ammotragus lervia), two European deers and three Hippo (Hippopotamus amphibius) beside one Nile lechwe (Kobus megaceros).. However, 123 fecal samples were collected from numerous bird species which consisted of two Ostriches (Struthio camelus), five Numida meleagris, twoIndian Peafowls (Pavo cristatus), two Emu (Dromaius novaehollandiae), 101 Pigeons (Columba livia domestica) and 11 Swan Goose (Anser cygnoides).

All fecal samples were collected with the help of the animal handlers. One visit per week during which freshly voided fecal samples was collected from the enclosure floor in the early morning directly after defecation in sterile cups and labeled by species. To obtain a representative sample from animals and birds housed in groups; the fecal sample was collected indiscriminately in various parts of the fencing and the number of collected samples was related to the number of the animals/ birds existing: a single pool was composed of five animals. Each fecal sample was placed in labeled clean plastic cups and preserved in an ice tank before transporting to the Parasitology Laboratory, Faculty of Veterinary medicine, Beni-Suef University for examination.

Human samples

Fresh stool samples were taken from seven zookeepers who had been in close contact with the examined animals and birds. Data concerning the consistency of stool samples, the presence or absence of gastrointestinal complaints, type and number served animals, were recorded. Samples were collected according to the WHO guidelines of fecal sample collection. Samples were labeled and sent to the laboratory for further parasitological examinations.

Examination techniques

Fecal samples were examined macroscopically to detect consistency, presence or absence of adult nematodes and proglottides of tapeworm. Meanwhile, microscopical examination was performed for detection of intestinal parasites. All fecal samples were processed and examined by direct wet mount preparation and salt flotation technique (Zajac and Conboy 2006; El-Ashram et al. 2019). Furthermore, the formol ether sedimentation technique was applied for each sample (Lee et al. 2010). To facilitate the identification of protozoan cysts and trophozoites; the lugol's iodine solution was applied (Arafa et al. 2012). Identification of parasites was based on the physical appearance of the fecal sample and microscopic examination according to the key provided by Soulsby (1986) and WHO (2020). Positive samples of Giardia cysts were preserved in alcohol and kept at 4 °C until used for DNA extraction (Sousa et al. 2006).

DNA extraction

Positive samples with Giardia cysts were identified microscopically in fecal and stool samples collected from zoo mammals and zoo keepers, then genetically by polymerase chain reaction targeting the glutamate dehydrogenase gene (gdh). Approximately 300 μl of fecal suspension was washed with distilled water three times to remove alcohol residue and then genomic DNA was extracted directly from feces using the DNA extraction kit (QIAamp DNA stool Mini Kit following the manufacturer's instruction (QIAGEN, Hilden, Germany). Briefly, 300 μl of fecal suspension was added to 1.4 ml buffer ASL, and then incubated at 70 °C for 5 min. Then, the samples were homogenized for 6 min. using the QIAGEN Tissue Lyser and then centrifuged at 14,000 rpm for 1 min. to pellet the stool. One Inhibit Ex tablet was added to 1.2 ml of the supernatant then vortexed and incubated for 1 min. at room temp. The samples were centrifuged at 14,000 rpm for 3 min. and 200 µl of the supernatant was added to 15 µl of proteinase K and 200 µl of lysis buffer AL and incubated at 70 °C for 10 min. After incubation, 200 µl of absolute ethanol was added to the lysate. The lysate was transported to the column, centrifuged at 14,000 rpm for 1 min., then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer AE provided in the kit (Babaei et al. 2008). The extracted DNA was stored in sterilized tubes at −20 °C for PCR reactions (Babaei et al. 2008).

Primers and PCR amplification

In the PCRreaction, A fragment of 432 bp was amplified using the forward primer (GDHiF) 5´-CAG TACAACTCTGCT CTCGG-3´ and the reverse primer (GDHiR), 5´-GTT GTC CTTGCA CATCTCC-3´ (Malekifard and Ahmadpour 2018). Primers were provided from Metabion (Germany) (Table 1). Programmable thermal cycler (Eppendorf, Hamburg, Germany) was done for PCR amplification. The amplification reaction was adjusted as follows, Primers were applied in a 25- µl reaction containing 12.5 µl of EmeraldAmp Max PCRMaster Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentrations, 6.5 µl of water, and 4 µl of DNAtemplate. The reaction was performed in a T3 Biometra thermal cycler (Eppendorf, Hamburg, Germany). Cycling parameters were 10 min at 94 °C (initial heat activation step), followed by 50 cycles of 35 s at 94 °C, 35 s at 61 °C and 50 s at 72 °C, with a final extension of 7 min at 72 °C. Positive and negative controls were involved in each PCR to confirm results. Cysts were used as the templates for the positive controls while distilled water for negative controls (Malekifard and Ahmadpour 2018).

Table 1.

Primers sequences, target genes, amplicon sizes and cycling conditions

Target agent	Primers sequences	Target gene	Amplified segment (bp)	Primary denaturation	Amplification (35 cycles)			Final extension	References
					Secondar denaturation	Annealing	Extension
Giardia intestinalis (Giardia duodenalis)	CAG TAC AAC TCT GCT CTC GG	gdh	432	94 °C 5 min	94 °C 30 s	61 °C 40 s	72 °C 45 s	72 °C 10 min	Malekifard and Ahmadpour (2018)
	GTT GTC CTT GCA CAT CTC C

Analysis of the PCR products

PCR products were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1 × TBE buffer at room temperature using gradients of 5 V/cm. For gel analysis, 15 µl of the products were loaded in each gel slot. Gel pilot 100 bp DNA Ladder (Qiagen, Germany, GmbH) was applied to define the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data were analyzed through computer software (Malekifard and Ahmadpour 2018).

Statistical analysis

All data were coded, entered, and analyzed using the statistical package SPSS version 22 (IBM Corp. Released 2013. IBM SPSS Statistics forWindows, Version 22.0. Armonk, NY: IBM Corp. USA). Results were statistically analyzed using a descriptive analysis and percentage for quantitative values to determine the prevalence of infection in examined animals and birds (Margolis et al. 1982). The prevalence for each parasite was calculated using the following formula:

$$\text{Prevalence}\left(\%\right)=\frac{\text{Number of infected individuals}\left(\text{n}\right)\times 100}{\text{Total number of sampled individuals}\left(\text{N}\right)}(\text{Julia et al}.\mathbf{2014})$$

Results

Fecal examination of 77 wild animals from various species at zoo Beni-Suef revealed that 48.05% were infected with different intestinal parasites; 11.69% of animals were positive with helminths, 27.27% with protozoa and 9.09% had mixed infections (Table 2). Generally, the prevalence of parasitic infection in primates was 75%, followed by carnivores 57.14% and herbivores 38.89%. Results indicated that protozoan infections (43.75%) were more common compared to helminth infections (6.25%) in primates, however the mixed infection was recorded only in 25% of the animals. The occurrence of helminths was 57.14% in carnivores with no detection of protozoa. However, for herbivores, a higher infection rate of protozoa (25.93%) was observed compared to helminths (11.69%) moreover mixed infection was 5.56% (Table 2).

Table 2.

The overall prevalence of intestinal parasitic infections among various animals in Zoo Beni-Suef

Animals	Examined animals	Helminth positive (%)	Protozoa positive (%)	Mixed infection (%)	Total (%)
Primates	16	1 (6.25)	7(43.75)	4 (25)	12 (75)
Carnivores	7	4 (57.14)	0	0	4 (57.14)
Herbivores	54	4 (7.40)	14 (25.93)	3 (5.56)	21(38.89)
Total	77	9(11.69)	21(27.27)	7(9.09)	37(48.05)

Data are presented as number of positive animals, with prevalence (%) in parentheses

Prevalence of mono and mixed infections in examined fecal samples of zoo animals

The overall prevalence of animals showed mono-infection was 37.66%. Identified parasites were Eimeria species with a prevalence of (11/77; 14.29%), Giardia spp. (7/77; 9.09%), Trichuris spp. (5/77; 6.49%), Ancylostomatidae (3/77; 3.89%), Toxocara spp. (2/77; 2.60%) and Spirometra spp. (1/77; 1.30%) (Supplementary Table 1, Fig. 1). Moreover, mixed infections were only detected in primates and herbivores with a prevalence of 9.09%; Trichuris spp. + Eimeria species was (3/77; 3.90%), Trichuris spp. + E. histolytica/dispar (2/77; 2.60%) and E. coli + Trichuris spp. (2/77; 2.60 (supplementary Table S2).

Fig. 1.

Different intestinal parasites recovered from non-human primates. a, b Trichuris spp. egg (Scale bar = 50 μm.); c Entamoeba coli cyst; d Giardia intestinalis cyst; e Giardia intestinalis trophozoite, f Entamoeba histolytica/dispar cyst; g Entamoeba histolytica/dispar cyst (Scale bar = 20 μm)

The prevalence of intestinal parasites in non-human primates

Giardia spp. showed the highest prevalence compared to other intestinal parasites in primates (43.75%). This is followed by mixed infections with E. histolytica/dispar + Trichuris spp. (12.5%) and E. coli + Trichuris spp. (12.5%) (Fig. 1).

The occurrence of Trichuris spp. was the lowest among parasites that were detected (6.25%). In both Grivet monkeys and Hamadryas baboons Giardia spp. recorded the highest infection rate; 40% and 75% respectively. Meanwhile, in Brown capuchin mixed infection with E. coli + Trichuris spp. was the highest (Table 3). Macroscopical examination of fecal samples from infected animals with E. histolytica/dispar and Giardia spp. appeared semi formed or diarrheic with offensive odor.

Table 3.

Prevalence (%) of intestinal parasites in non-human primates at zoo Beni-Suef

Common name	Scientific name	No. of examined	Giardia spp. (%)	Entamoeba histolytica/dispar + Trichuris spp. (%)	E. coli + Trichuris spp. (%)	Trichuris spp. (%)	Total (%)
Grivet monkeys	Cholorocebs aethiops	10	4 (40)	2 (20)	–	–	6 (60)
Brown capuchin	Cebus apella	2	–	–	2 (100)	–	2 (100)
Hamadryas baboons	Papio hamadryas	4	3 (75)			1(25)	4 (100)
Total		16	7 (43.75)	2 (12.5)	2 (12.5)	1(6.25)	12 (75)

No.: Number of animals, Data are presented as number of positive animals, with prevalence (%) in parentheses

The prevalence of intestinal parasites in carnivores and herbivores

Spirometra spp., and Ancylostomatidae were reported in the examined carnivores. In African lions Anclystomatidae. recorded the highest infection rate (40%) followed by Spirometra spp. (20%). Moreover, Ancylostomatidae had the infection rate of 50% in Hyena (Table 4, Fig. 2).

Table 4.

Prevalence (%) of intestinal parasites in carnivores and herbivores at zoo Beni-Suef

Carnivores		No. of animals examined	No. of animals infected	Prevalence (%)	Animals showed single infection (%)	Animals showed mixed infection (%)	Parasites detected (%)
Common name	Scientific name						Spirometra spp.(%)	Trichuris spp. (%)	Toxocara spp. (%)	Ancylostomatidae (%)	Blantidium coli. (%)	Eimeria spp. (%)
African lion	Panther Leo	5	3	60	3	0	1 (20)			2(40)
Hyena	Crocuta crocuta	2	1	50	1	0				1 (50)
Total		7	4	57.14	4	0	1 (14.29)			3 (42.86)
Herbivores
Lamma	Lama glama	1	1	100	1	0					1(100)
Pony	Shetland pony	1	1	100	1	0			1 (100)
Nile lechwe	Kobus megaceros	1	1	100	1	0			1 (100)
Mountain goats	Capra aergagrus	34	13	38.24	11	2		3 (8.82)			3 (8.82)	7 (20.59)
Barbary sheep	Ammotragus lervia	12	5	41.66	4	1		1 (8.33)				4 (33.33)
Hippo	Hippopotamus amphibious	3	0	0	0	0	-	-	-	-	-
Deer	European deer	2	0	0	0	0	-	-	-	-	-
Total (%)		54	21	38.89	18 (33.33)	3 (5.56)	-	4(7.41)	2 (3.70)		4 (7.41)	11 (20.37)

No.: Number of animals

Fig. 2.

Intestinal parasites diagnosed in fecal samples from carnivores and herbivores. a Toxocara spp. egg; b Sublura brumbti egg; c Capillaria spp. egg; d Spirometra spp. egg; e Blantidium coli cyst; f, g Un sporualted oocyst Eimeia spp.; h Ancylostomatidae egg; i Isospora spp. egg (Scale bar = 50 μm)

The prevalence of Eimeria spp. (20.37%) was the highest among different types of intestinal parasites found in herbivores followed by B. coli and Trichuris spp. (7.41%). Furthermore, Toxocara spp. (3.70%) recorded the lowest prevalence of infection. In Mountain goats Eimeria spp. recorded the highest infection rate 20.59% followed by B. coil and Trichuris spp. 8.82%. Meanwhile, the prevalence of Trichuris spp. and Ancylostomatidae were the lowest (5.88%) among the intestinal parasites reported in Mountain goats. Strikingly, Eimeria spp. and Trichuris spp. infections were reported in Barbary sheep with prevalence of 33.33% and 8.33% respectively. In addition, B. coli was the only protozoa detected in Lama glama with a prevalence of 100%. Meanwhile, the infection rate of Toxocara spp. in Shetland pony and Kobus megaceros was 100%. Oppositely, fecal examination of Hippo and Deer showed a negative result for intestinal parasites (Table 4).

The overall prevalence of intestinal parasites in examined zoo birds

Out of 123 fecal samples were examined, 27 were found to be infected with different types of intestinal parasites with a prevalence of 21.95%. Mixed infection was recorded in all zoo birds. Infection with Eimeria spp. and Sublura brumpti was the highest with a prevalence of 10.57% followed by Eimeria spp. and Giardia spp. 4.07%. Also, the infection rate of Isospora spp. and Eimeria spp. was 4.07%. Meanwhile, the prevalence of Eimeria spp. and Capillaria spp. was the lowest 3.25%. In pigeons infection with Eimeria spp. and Sublura brumpti were detected. However, Numida meleagris showed mixed infection with Eimeria spp. and Giardia spp. In Emu and Peafowl infection with Eimeria spp. and Capillaria spp. were recorded but Swan Goose infected with Eimeria spp. and Isospora spp. Oppositely, Ostrich was free from gastrointestinal parasites (Table 5).

Table 5.

Enteric parasites diagnosed in fecal samples of birds at Zoo Beni-Suef

Animal species (Zoo birds)		No. of examined birds	Positive birds (%)	Parasites detected (Mixed infections) (%)
Common name	Scientific name			Eimeria spp. + Giardia spp. (%)	Eimeria spp. + Capillaria spp.(%)	Eimeria spp. + Isospora spp.(%)	Eimeria spp. + Sublura brumpti (%)_
Numida meleagris	Numida meleagris	5	5 (100)	5(100)	-	-	-
Emu	Dromaius novaehollandiae	2	2 (100)	-	2(100%)	-	-
Swan Goose	Anser cygnoides	11	5 (45.45)	-	-	5(45.45)	-
Ostrich	Struthio camelus	2	0	Nil	Nil	Nil	Nil
Peafowl	Pavo cristatus	2	2 (100)	-	2(100)	-	-
Pigeon	Columba livia domestica	101	13 (12.87)	-	-	-	13(12.87)
	Total (%)	123	27(21.95)	5 (4.07)	4(3.25)	5 (4.07)	13(10.57)

No.: Number of animals, Data are presented as number of positive animals, with prevalence (%) in parentheses

Microscopical findings of zookeeper's stool samples

In zookeepers, Giardia spp. recorded the highest infection rate 42.85% followed by Entamoeba histolytica/ dispar with a prevalence of 28.57%. Two workers serving non-human primates and one serving zoo birds revealed infection with Giardia intestinalis. On the other hand, infection with Entamoeba histolytica/dispar was only recorded in workers who serving non-human primates only (Supplementary Table S3).

The PCR amplification

The DNAs were successfully extracted from all positive samples for Giardia spp. cysts (humans and non-human primates) and the PCR fragments were successfully visualized on gel electrophoresis. The gdh gene of G. duodenalis was successfully amplified from seven samples of zoo animals (9.09%) and three samples of zookeepers (42.85%); A 432 bp fragment of gdh gene was amplified in the PCR using GDHiF and GDHiR primers (Fig. 3).

Fig. 3.

Electrophoretic separation of PCR products from DNA targeting the gdh gene of G. duodenalis on an ethidium bromide stained 1.50% agarose gel at 432 bp. Lane L: 100 bp gene ruler (Fermentas). Lane Pos: Positive control. Lane Neg: Negative control. Lane 1: The PCR products from positive human sample (432 bp fragment). Lane 2: The PCR products from positive Grivet monkey (Non-human primates). Lane 3: The PCR products from positive Hamadryas baboons (432 bp fragment)

Discussion

Zoo mammals and birds (wild and domestics) have an essential role as a host and reservoir for many zoonotic parasites. The current study revealed that the overall prevalence of intestinal parasitic infection among various zoo mammal was 48.05% with 11.69% positive with helminths, 27.27% with protozoa and 9.09% had mixed infection. Results indicated that protozoan infection was more common than that of helminth among zoomammals. These results are not in agreement with those recorded by Lim et al. (2008) who detected a higher prevalence of helminths 34.5% than protozoa 21.8% in Malaysia. Such variations could be due to climatic, seasonal and geographical differences and husbandry activities in different areas of the studied animals.

In the current investigation, the most common intestinal parasites among non-human primates were Giardia intestinalis, Entamoeba histolytica /dispar and Trichuris trichura infections. These parasites have a direct life cycle, which explained their currency and increases the zoonotic potential for individuals at risk. Similar findings were reported by Thomas et al. (2005) in Uganda, Arafa et al. (2012) in Beni-Suef zoo, Egypt and Naoki et al. (2012) from wild Japanese Macaques. Our study revealed that a higher prevalence of protozoa (56.25%) compared to helminths (6.25%) in primates. These findings are closely related to that reported by Lim et al. (2008) who detected a higher occurrence of protozoa (35.4%) compared to helminths (19.1%) in Malaysia. Oppositely, observation in a zoological garden at Kenya by Munene et al. (1998) found a higher prevalence of helminths (64.4%) and a lower of protozoa (17.1%). Such differences may be due to seasonal, climatic, and geographic conditions and animal management in various regions of the diagnosed animals. Herein, it was observed that the animals infected with Giardia intestinalis suffered from persistent diarrhea, abdominal pain and weight loss. That result comes in contact with the observations recorded by Lee et al. (2010) and Arafa et al. (2012) who observed that Giardia spp. caused diarrhea in monkeys.

Fecal examination of carnivores revealed that 57.14% of animals were positive for different intestinal parasites. The most common parasites were Anclystomatidae spp. with the highest prevalence (42.86%) followed by Spirometra spp. (14.29%). Similar findings were reported by Abe and Yasukawa (1996) and Lim et al. (2008) who recorded that 64.3% of feline were positive for T. cati. Furthermore, previous literature detected that tapeworms were common among zoo animals by Chauhan et al. (1973) and Sen Gupta (1974).

Regarding herbivores, 38.89% of animals were found to be infected with different intestinal parasites. These findings are slightly similar to those obtained by Chakraborty and Islam (1996) who observed that the prevalence of parasitic infection was 40.4% in Dhaka Zoo. However, the findings of the present study do not support previous literature that recorded a higher prevalence of intestinal parasites in herbivores as 60.7% by Parasani et al. (2001) and 56.3% by Lim et al. (2008). These discrepancies in the infection rates might be due to geographic factors, husbandry practice and the animal's source of feed that affects the prevalence. The study revealed a higher occurrence of protozoa (25.93%) was observed compared to helminths (11.69%). Such results come in line with the earlier reports of Opara et al. (2010) who recorded a higher prevalence of protozoa (82.2%) than helminths (17.8%). Contrariwise, in Rajkot Municipal Corporation zoo Parasani et al. (2001) revealed that 50% of the animals were positive for helminth infection and 18.8% for protozoa. This could be qualified to the source of feeds of the animal which influences the prevalence. The current investigation revealed that18.52% of animals infected with Eimeria spp. followed by B. coli 7.41%, Hookworm 5.56%, Trichuris spp. 5.56% and Toxocara spp. 1.85%. These results agree with previous studies by (Geraghty et al. 1982 and Lim et al. 2008). On the contrary, our results differ from Ruta et al. (2009) who found Strongyloides sp. (73.3%) and Paramphistomum sp. (63.0%) in red deer and Opara et al. (2010) who observed Ascaris sp. and Fasciola sp. in different zoo animals. This difference might be due to the location of animal cages, availability of intermediate hosts near the cages and the source of feeds.

Moreover, mixed infections in Mountain goats and Barbary sheep were recorded with a prevalence of 5.56%. This finding was lower than that reported by Rahman et al. (2014) 42.3%. The presence of mixed infection in this study might be due to the presence of different aged animals in the same cages, feeding management and improper fecal disposable.

Out of 123 birds were examined, 27 birds were found to be infected with different types of gastrointestinal parasites with a prevalence of 21.95%; the identified parasites were Giardia spp. Eimeria spp., Capillaria spp., Isospora spp. and Sublura brumbti. The obtained findings are in accordance with Otegbade and Morenikeji (2014) in Nigeria who recorded an overall prevalence was 21.9%. Our results were lower than that recorded by Opara et al. (2010) and Akinboye et al. (2010) who stated prevalence rates of 76.6% and 61.5% in Nekede and U.I Zoos, respectively. Oppositely, Ajibade et al. (2010) recorded no infection of birds in OAU and U.I Zoos in Nigeria. This variation could be qualified to the husbandry dependent factors such as housing and feeding, conflict in the treatment program, or the existence of favorable climatic conditions (Magona and Musisi 1999).

Regarding the fecal examination of zookeepers; it revealed that Giardia intestinalis recorded the highest infection rate (42.85%) followed by Entamoeba histolytica /dispar (28.57%). Both parasites have a direct life cycle so easily to be transmitted through contaminated food, water or soil (Akinboye et al. 2010). As more, Akinboye et al. (2010) detected Giardia spp. and Entamoeba histolytica in 22.2% and 11.1% respectively in stool samples of zoo workers at the University of Ibadan Zoological Garden, Nigeria. These parasites are of zoonotic importance and constitute a health problem to humans, mainly animal handlers who come in contact with these animals. Although, zoo workers facing a great risk of contracting zoonotic pathogens from zoo animals also they act as a vector of transmission of such parasites between animals (Mir et al. 2016; Panayotov-Pencheva 2013; Otegbade and Morenikeji 2014).

In the present investigation, a variety of zoo animal species and zookeepers were tested for the presence of Giardia cysts in fecal and stool samples, and an overall prevalence of 9.09% (7/77) was estimated in zoo animals and 42.85% (3/7) in zookeepers. Several genetic loci have recently been used to classify Giardia isolates at genotype and subtype levels, most frequently containing the small subunit ribosomal RNA (SSUrRNA), triosephosphate (tpi), and glutamate dehydrogenase (gdh). Glutamate dehydrogenase (gdh) is one of the most important genetic tools for the genetic identification of Giardia duodenalis (Thompson 2004; Siripattanapipong et al. 2011). Positive samples with Giardia cysts were identified microscopically in fecal and stool samples, then genetically by polymerase chain reaction targeting the glutamate dehydrogenase gene (gdh). The gdh gene of G. doudenalis was successfully amplified from seven samples of zoo animals (primates) and three samples of zookeepers. A 432 bp fragment of gdh gene was amplified in the PCR using GDHiF and GDHiR primers according to Malekifard and Ahmadpour (2018). Previous studies by Bertrand et al. (2005) found that 21(81%) samples were positive when gdh gene was targeted. Moreover, David et al. (2011) revealed that 95.7% of microscopically positive Giardia samples were positive for gdh genes. Furthermore, Faria et al. (2016) found that 41 (63.1%) out of 65 positive samples for Giardia were positively amplified by PCR targeting the gdh gene. Finally, Chanu et al. (2018) established that out of 20 samples, 13 (65%) were positive for PCR assay targeting gdh gene.

Conclusion

This study highlighted the importance of controlling parasite infection rates in zoological gardens. The intestinal parasites detected in this investigation are recognized to be of human pathogenic importance as a potential source for zoonotic transmission between animals and humans especially among zookeepers. An Effective control measure is necessary to protect the health of Zoo animals.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

AAK designed the study, analyzed and processed data, examination and identification of samples. GKA helped in samples collection, data evaluation and edited in the manuscript.

Funding

None.

Compliance with ethical standards

Conflict of interest

Authors declare that they have no conflict of interest.

Ethical statement

This study was conducted according to the ethical standards of Faculty of Veterinary Medicine, Beni-Suef University, Egypt and approved by the Institutional Animal Care and Use Committee of Beni-Suef University (2019-BSUV-39).

Informed consent

The zookeepers involved in this investigation consented to facilitate this study.