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Entamoeba infections in different populations of dogs in an endemic area of Lahore, Pakistan
Veterinary Parasitology 207 (2015) 216–219
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Entamoeba infections in different populations of dogs in an endemic area of Lahore, Pakistan Muhammad Azhar Alam a , Azhar Maqbool a , Muhammad Mudasser Nazir b,∗ , Muhammad Lateef a , Muhammad Sarwar Khan c , David S. Lindsay d a
Department of Parasitology, University of Veterinary and Animal Sciences, Lahore 54600, Pakistan Department of Pathobiology, Faculty of Veterinary Sciences, B.Z. University, Multan 60800, Pakistan Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, Lahore 54600, Pakistan d Department of Biomedical Sciences and Pathobiology, Virginia–Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, United States b c
a r t i c l e
i n f o
Article history: Received 26 June 2014 Received in revised form 28 November 2014 Accepted 2 December 2014 Keywords: Entamoeba histolytica Dog Cyst Household Pathogen
a b s t r a c t Entamoeba histolytica, a protozoan parasite that affects humans and other primates all over the world. It is a common waterborne pathogen in endemic areas that have fecal oral transmission cycle. The aim of the present study was to examine the prevalence of E. histolytica and other Entamoeba species cysts in three different dog populations. Fecal samples from 600 dogs were collected and processed to detect Entamoeba cysts using the triple fecal test (light microscopy) and fecal antigens of E. histolytica were detected using a fecal antigen ELISA (TechLab E. histolytica II). Because it is impossible to differentiate E. histolytica from Entamoeba dispar and E. moshkovskii, using light microscopy we referred to all cysts morphologically consistent with E. histolytica as E. histolytica/dispar/moskovskii to reflect this uncertainty. Samples from 197 household dogs without clinical signs, 122 samples from household dogs exhibiting clinical signs of diarrhea, dysentery and vomiting and 281 stray dogs with no specific clinical signs were examined. Entamoeba histolytica-like cysts were observed in 94 (15.6%, 95% CI = ±3.88) by triple fecal test microscopy and E. histolytica antigens were demonstrated in 66 (11%, 95% CI = ±4.41) by fecal antigen ELISA in 600 fecal samples. Significant differences (P ≤ 0.05) in prevalence were found between the three populations. Twenty (10.1%, 95% CI = ±7.86) and 11 (5.6%, 95% CI = ±7.70) of 197 fecal samples from household dogs without clinical signs were positive by microscopy and by antigen ELISA, respectively. Twenty-nine (23.8%, 95% CI = ±6.58) and 23 (18.8%, 95% CI = ±7.81) of 122 the fecal samples from household dogs with clinical signs were positive by microscopy and by antigen ELISA, respectively. Forty-five (16.01%, 95% CI = ±5.62) and 32 (11.3%, 95% CI = ±6.38) of 281 fecal samples from stray dogs were positive by microscopy and by fecal antigen ELISA, respectively. Dogs from the youngest age group (6 months to 1 year) were more likely to be E. histolytica antigen positive than were dogs from the other two older age groups, with a significant difference (P ≤ 0.05) between all age groups. Statistically, no significant (P ≥ 0.05) difference of prevalence was seen in male and female dogs. The local dogs had the highest prevalence rate of E. histolytica antigens (36 of 246, 14.2%, 95% CI = ±6.32) followed by imported breeds (11 of 115, 9.5%, 95% CI = ±10.4) and crossbred (19 of 239, 8.3%, 95% CI = ±7.47), indicating a significant (P ≤ 0.05) trend of positivity between various breeds
∗ Corresponding author. E-mail address: [email protected] (M.M. Nazir). http://dx.doi.org/10.1016/j.vetpar.2014.12.001 0304-4017/© 2014 Elsevier B.V. All rights reserved.
M.A. Alam et al. / Veterinary Parasitology 207 (2015) 216–219
217
of dogs. These findings suggest that dogs may play an important role in the epidemiology of this pathogen. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Amebic dysentery is a disease of man and animals caused by the intestinal protozoan parasite Entamoeba histolytica. It is characterized by severe to moderate gastroenteritis. In advance cases, infection may lead to anemia and liver abscess that can spread to other organs particularly lungs and brain (Roberts et al., 1973; Haque et al., 2003). Globally, amebiasis is the third most prevalent cause of human mortality after malaria and schistomiasis. It is responsible for 40,000 deaths per annum (Sebastiaan et al., 2007). It is estimated that 10% of the world’s population are effected with amoebe predominantly in developing countries in tropical and subtropical areas (Ravdin, 2000; Stanly, 2003). The major source of transmission is untreated drinking water and food contaminated by E. histolytica cysts (Bruckner, 1992). The genus Entamoeba consists of many species but E. histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanni are commonly found in the intestinal lumen of humans and other mammals (Fotedar et al., 2007). E. histolytica was once considered to be a single species, but advance molecular techniques and isoenzyme studies indicated that it be reclassified into two morphologically identical species: the pathogenic E. histolytica and the non-pathogenic E. dispar that are morphologically identical but genetically and biochemically different (Stark et al., 2007). The diagnosis of Entamoeba infection initially relies on fecal examination but due to morphological resemblance of these Entamoeba species it cannot be made by routine staining methods. Pathogenic E. histolytica from identical non-pathogenic E. dispar and E. moshkovskii can only be differentiated by advance methods such as Entamoeba antigen ELISA, DNA detection, PCR and isoenzymes analysis (Markell et al., 1999; Tanyuksel and Petri, 2003; Ximénez and Nosaki, 2014). Dogs are important in the developed and developing countries. Their role as companion and service animals affords them an important position in the human world compared to other domestic animals (Jane, 1996). Because of this association the role of dogs as sources of parasites infective for their human owners is important (Lorenzini et al., 2007). Because of the importance to human health and limited current information on prevalence, we conducted the current study to determine the prevalence of Entamoeba infection in dogs in an endemic area of Lahore, Pakistan.
7,566,000 individuals. The study group was designed by clustered sampling and size of the sample was calculated by online survey system software (sample size calculator). Three dogs populations of different breeds and age groups were evaluated to detect E. histolytica/dispar/moskovskii cysts using light microscopy and for fecal antigens of E. histolytica. A total of 600 fecal samples were obtained from three groups of dogs, the first group consisted of 197 household dogs without clinical signs, group 2 contained 122 household dogs exhibiting clinical signs of diarrhea, dysentery and vomiting were brought to 9 veterinary clinics at various locations in Lahore, Pakistan. Group 3 consisted of 281 stray dogs collected from the city. The study was approved by the Directorate of advance Studies and Research Board, UVAS and Department of Epidemics and Public Health, Lahore. Fecal samples were obtained from the rectum of dogs by using disposable gloves and placed into clean and dry universal glass bottles and taken to the Diagnostic Parasitology Laboratory, Department of Parasitology, University of Veterinary and Animal Sciences, Lahore for further processing. Data regarding epidemiological information on dogs age, sex and breed were obtained from owners, practitioners and researchers at the time of sampling. The date of sample collection was also recorded and used to evaluate the influence on season of the year with parasite prevalence. Information about clinical symptoms i.e. dogs with diarrhea, dysentery, fever and vomiting followed by weight loss in some cases were also recorded on a separate data sheet at the time of sampling. 2.2. Laboratory diagnosis
2. Materials and methods
About 5 g of fecal sample was fixed in methanol and examined by the triple fecal test (Van Gool et al., 2003) to detect fecal cysts with morphology consistent with E. histolytica (referred to as E. histolytica/dispar/moskovskii). A small portion of fecal sample was not fixed but kept at 4 ◦ C until examined for fecal antigens of E. histolytica using a commercially available fecal antigen ELISA kit (Tech Lab, Blacksburg, VA, USA). The samples were processed according to the manufacturer’s instructions. Positive and negative controls were provided with the kit. Because cyst morphology cannot be used to definitively identify E. histolytica, we refer to samples positive for cysts by the triple test as being E. histolytica/dispar/moskovskii. Only samples positive by antigen ELISA were declared to be true E. histolytica positives (Table 1). Triple fecal test positive samples were stained with permanent stain (chlorazole black stain). Samples evaluated by using fecal antigen ELISA were read using an ELISA plate reader (Thermolectron, Finland) operating at 450 nm wavelength.
2.1. Dog populations and sample collection
2.3. Dogs breed and sex
Lahore is the second largest metropolitan city in Pakistan with an area of 1172 km2 and population of
The dogs were classified into exotic breeds, local breeds and crossbreeds (cross between local and exotic breeds).
218
M.A. Alam et al. / Veterinary Parasitology 207 (2015) 216–219
Table 1 Prevalence of Entamoeba histolytica/dispar/moskovskii cysts and E. histolytica antigen positive samples among dog populations. Characteristics
No. examined (N)
Category
Triple fecal test
ELISA
Positive (n)
% (95% CI)
P value
Positive (n)
% (95% CI)
0.011
11
5.58 (±7.70)
23
18.85 (±7.81)
P value
Household dogs without clinical signs Household dogs with clinical signs Stray dogs
197
20
10.15 (±7.86)
122
29
23.77 (±6.58)
281
45
16.01 (5.62)
32
11.38 (±6.38)
Sex
Male Female
362 238
58 36
16.0 (±4.95) 15.12 (±6.26)
0.768
45 21
12.4 (±5.47) 8.8 (±7.40)
0.167
Age group
6 months to 1 year 1–6 year >6 year
145 170 285
36 28 30
24.82 (±5.79) 16.47 (±7.12) 10.52 (±6.48)
0.001
27 19 20
18.6 (±7.22) 11.2 (±8.24) 7.0 (±7.06)
0.0012
Breed
Exotic Local Crossbred
115 246 239
16 49 29
13.9 (±9.33) 19.9 (±5.31) 12.1 (±6.79)
0.000
11 36 19
9.5 (±10.4) 14.2 (±6.32) 8.3 (±7.47)
0.001
Total
600
94
15.66 (±3.88)
66
11.0(±4.41)
Dogs group
Dog breed was defined based on pedigree standards of the Kennel Club of Pakistan. A dog was placed in the crossbred group if it possessed the phenotype of two distinct breeds and all native dog breeds were compiled in local breeds. Each dog’s sex was recorded as male or female despite of if the animal had been neutered or spayed and the dogs were classified into 3 different age groups (Table 1). Age groups were 6 months to 1 year (N = 145), 1–6 years (N = 170), greater than 6 years (N = 285). 2.4. Statistical examination The percentage estimates 95% confidence level was calculated by Microsoft Excel 2010 software and the values between different populations are compared by Pearson’s x2 test with respect to age, breed and sex using (SPSS for Windows, version 20.0; SPSS, Chicago, IL), and P ≤ 0.05 was considered as significant prior to conduct of the test. 3. Results The results showed that out of 600 fecal samples examined 94 (15.6%, 95% CI = ±3.88) were positive for E. histolytica/dispar/moskovskii cysts by microscopy while 66 (11%, 95% CI = ±4.41) of samples were found positive by antigen ELISA (Table 1). Prevalence rates of E. histolytica/dispar/moskovskii cysts (29/122, 23.77%, 95% CI = ±6.58) and E. histolytica antigens (23/122, 18.85%, 95% CI = ±7.81) were highest in household dogs with clinical signs, stray dogs were the next group having high prevalence of E. histolytica/dispar/moskovskii cysts (45/281, 16.01%, 95% CI = ±5.62) and E. histolytica antigens (32 of 281, 11.38%, 95% CI = ±6.38), whereas the household dogs without clinical signs had the lowest prevalence of E. histolytica/dispar/moskovskii cysts (20/197, 10.15%, 95% CI = ±7.86) and E. histolytica antigens (11 of 197, 5.58%, 95% CI = ±7.70). The prevalence rates were significantly (P ≤ 0.05) different within three populations of dogs both by microscopy and antigen ELISA (Table 1). Likewise
0.012
prevalence rates were significantly (P ≤ 0.05) different between dogs with and without clinical signs (Table 1). Our results demonstrated that 17 of 94 (18%) samples positive by light microscopy were negative by fecal antigen ELISA. Five (17.2%) samples from household dogs with clinical signs, three (15%) from household dogs without clinical signs and 9 (19.4%) from stray dogs were triple fecal test positive samples that were negative for fecal antigen ELISA. These findings represent the potential prevalence of E. dispar and E. moshkovskii in dog’s population. Male dogs were more likely to be positive for E. histolytica/dispar/moskovskii cysts (58/362, 16%, 95% CI = ±4.95) and E. histolytica antigens (45 of 362, 12.4%, 95% CI = ±5.47), than female dogs which had a prevalence of E. histolytica/dispar/moskovskii cysts (36/238, 15.12%, 95% CI = ±6.26) and E. histolytica antigens (21 of 238, 8.8%, 95% CI = ±7.40). This was not statistically (P≥ 0.05) significant (Table 1). The prevalence of dogs that were positive for E. histolytica/dispar/moskovskii and for E. histolytica antigens by ELISA was highest in the youngest age group (6 months to 1 year) (Table 1). Prevalence of infection based on age group was significantly (P ≤ 0.05) different using either triple fecal test or E. histolytica antigen ELISA. The prevalence of E. histolytica/dispar/moskovskii cysts and E. histolytica fecal antigen positive samples was interlinked with season of the year. Most of the positive cases were observed during the summer season and fewest in the winter months. The local dogs had the highest prevalence of E. histolytica/dispar/moskovskii cysts (49/246, 19.9%, 95% CI = ±5.31) and E. histolytica antigen (36 of 249, 14.2%, 95% CI = ±6.32) followed by exotic and crossbred (Table 1). However, no significant (P ≤ 0.05) difference of prevalence among different dog breeds was observed using both microscopy and antigen ELISA. 4. Discussion Amoebic dysentery, an intestinal disease caused by infection with the protozoan parasite E. histolytica, is an
M.A. Alam et al. / Veterinary Parasitology 207 (2015) 216–219
important disease of man and animals throughout many areas of the world. In the present study a high prevalence of E. histolytica/dispar/moskovskii cysts were recorded in dogs by triple fecal test and relatively low prevalence rate of true E. histolytica infection was observed in dogs based on fecal antigen ELISA. However, the difference of prevalence recorded by two tests was statistically significant (P ≤ 0.05). As triple fecal test cannot differentiate between potentially pathogenic E. histolytica and non-pathogenic but morphologically indistinguishable E. dispar or E. moskovski so all cysts viewed by light microscopy were called E. histolytica/dispar/moskovskii. The fecal antigen ELISA is specific for E. histolytica and does not cross react with E. dispar and is considered as true E. histolytica infection in the current report. The prevalence rate is analogous to the formerly reported study based on microscopy with high prevalence rate in dogs (Johnson et al., 2010). The current findings of E. histolytica antigen positive dog samples gives us a clue that dogs can play an important role in the epidemiology of Entamoeba infection in endemic areas. The highest prevalence rate of infection was observed in symptomatic household dogs as compared to stray dogs and asymptomatic household dogs (Table 1). The higher prevalence of Entamoeba infection in household symptomatic dogs may be due to close association of dogs and humans. Ingestion of contaminated food and water may be a possible source of transmission of infection between dogs and humans. The sex wise prevalence of this disease was observed as, male dogs had higher prevalence than the female dogs. However, in an earlier study, female dogs had higher prevalence than male dogs (Mahfooz et al., 2014). Our results confirmed that the prevalence of infection with E. histolytica/dispar/moskovskii cysts and E. histolytica fecal antigens decreased with the dog’s age (Table 1) and it was highest in the youngest age group as previously, reported by others (Sarger et al., 2006b, Lorenzini et al., 2007; Martinez-Moreno et al., 2007). The highest prevalence rate of infection in young dogs might be due to immuno incompetence as a result of reduced level of passive immunity received from the mothers (OliveriaSequeira et al., 2002). We found a significant (P ≤ 0.05) difference in prevalence of Entamoeba infection among different breeds of dogs and in local dogs compared to other groups of dogs. This corresponds to previous observations made by Johnson et al. (2010) and Mahfooz et al. (2014). These reports should be interpreted carefully because housing conditions may influence transmission of Entamoeba species in dogs.
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Acknowledgments The current study was supported by a scholarship to M.A.A. from the Higher Education Commission, Islamabad, Pakistan. The authors would like to thank Dr. Atta Subhani, Muhammad Asif Ali, Dr. M. Oneeb, Dr Tanveer ul Haq, Dr. Ahsan Mustafa, Noman Nazir, and Muhammad Kashif Nazir for their moral enthusiasm and technical assistance during this study. References Bruckner, D.A., 1992. Amebiasis. Clin. Microbiol. Rev. 5, 356–369. Fotedar, R., Stark, D., Beebe, N., Marriott, D., Ellis, J., Harkness, J., 2007. PCR detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from Sydney, Australia. J. Clin. Microbiol. 45, 1035–1037. Haque, R., Huston, C.D., Hughes, M., Houpt, E., Petri, W.A., 2003. Amebiasis. N. Engl. J. Med. 348, 1565–1573. Jane, E., 1996. Collins Gem Dogs Photoguide, first ed., pp. 5–6. Johnson, O., Adejinmi, Joseph, O., 2010. Prevalence of intestinal protozoan parasites of dogs in Ibadan, south western Nigeria. J. Anim. Plant Sci. 7, 783–788. Lorenzini, G., Tasca, T., De Carli, G.A., 2007. Prevalence of intestinal parasites in dogs and cats under veterinary care in Porto Ategre, Rio Grande do Sul, Brazil. Braz. J. Vet. Res. Anim. Sci. 44, 137–145. Mahfooz, A., lqbal, M., Ahmad, T., Khan, M., Iqbal, A., Saleem, I., 2014. Amoebiasis: prevalence, treatment with metronidazole and nalidixic acid in dogs. Sch. Ad. Anim. Vet. Res. 1, 13–15. Markell, E.K., John, D.T., Krotoski, W.A., 1999. Medical Parasitology, eighth ed. Saunders Company, Mexico, pp. 24–89. Martinez-Moreno, F.J., Hernandez, S., Lopez, C.E., Becerra, C., Acosta, I., Martinez Mareio, A., 2007. Estimation of canine intestinal parasites in Cordoba (Spain) and their risk to public health. Vet. Parasitol. 143, 7–13. Oliveria-Sequeira, T.G., Amarante, A., Ferrari, T.B., Nunes, L.C., 2002. Prevalence of intestinal parasites in dogs from Sao Panto State, Brazil. Vet. Parasitol. 103, 19–27. Ravdin, J.I., 2000. Amoebiasis. Imperial College Press, London, pp. 47–66. Roberts, E.D., Williams, J.C., Piie, G., 1973. Naturally occurring gastric amebiasis in the wallaroo. Vet. Pathol. 10, 323–329. Sarger, H., Steiner, M.C., Multer, N., Staubli, D., Esposito, M., Schares, G., Hassig, M., Stark, K., Gottsterin, B., 2006b. Incidence of Neospora caninum and other intestinal protozoan parasites in populations of Swiss dogs. Vet. Parasitol. 139, 84–92. Sebastiaan, J., Van Hal, J.V., Stark, D.J., Fotedar, R., Marriott, D., John, T., Ellis, J.T., Harkness, J.L., 2007. Amoebiasis: current status in Australia. Med. J. Aust. 186, 412–416. Stanly, S.L., 2003. Amoebiasis. Lancet 361, 1025–1034. Stark, D., Fotedar, R., Van Hal, S., Beebe, N., Marriott, D., Ellis, J.T., Harkness, J., 2007. Prevalence of enteric protozoa in human immunodeficiency virus (HIV)-positive and HIV-negative men who have sex with men from Sydney. Aust. Am. J. Trop. Med. Hyg. 76, 49–552. Tanyuksel, M., Petri, W.A., 2003. Laboratory diagnosis of amoebiasis. Clin. Microbiol. Rev. 16, 713–729. Van Gool, T., Weijts, R., Lommerse, E., Mank, T.G., 2003. Triple faeces test: an effective tool for detection of intestinal parasites in routine clinical practice. Eur. J. Clin. Microbiol. Inf. Dis. 22, 284–290. Ximénez, C., Nosaki, T., 2014. Metabolomic analysis of Entamoeba: applications and implications. Curr. Opin. Microbiol. 20, 118–124.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Entamoeba infections in different populations of dogs in an endemic area of Lahore, Pakistan Muhammad Azhar Alam a , Azhar Maqbool a , Muhammad Mudasser Nazir b,∗ , Muhammad Lateef a , Muhammad Sarwar Khan c , David S. Lindsay d a
Department of Parasitology, University of Veterinary and Animal Sciences, Lahore 54600, Pakistan Department of Pathobiology, Faculty of Veterinary Sciences, B.Z. University, Multan 60800, Pakistan Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, Lahore 54600, Pakistan d Department of Biomedical Sciences and Pathobiology, Virginia–Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, United States b c
a r t i c l e
i n f o
Article history: Received 26 June 2014 Received in revised form 28 November 2014 Accepted 2 December 2014 Keywords: Entamoeba histolytica Dog Cyst Household Pathogen
a b s t r a c t Entamoeba histolytica, a protozoan parasite that affects humans and other primates all over the world. It is a common waterborne pathogen in endemic areas that have fecal oral transmission cycle. The aim of the present study was to examine the prevalence of E. histolytica and other Entamoeba species cysts in three different dog populations. Fecal samples from 600 dogs were collected and processed to detect Entamoeba cysts using the triple fecal test (light microscopy) and fecal antigens of E. histolytica were detected using a fecal antigen ELISA (TechLab E. histolytica II). Because it is impossible to differentiate E. histolytica from Entamoeba dispar and E. moshkovskii, using light microscopy we referred to all cysts morphologically consistent with E. histolytica as E. histolytica/dispar/moskovskii to reflect this uncertainty. Samples from 197 household dogs without clinical signs, 122 samples from household dogs exhibiting clinical signs of diarrhea, dysentery and vomiting and 281 stray dogs with no specific clinical signs were examined. Entamoeba histolytica-like cysts were observed in 94 (15.6%, 95% CI = ±3.88) by triple fecal test microscopy and E. histolytica antigens were demonstrated in 66 (11%, 95% CI = ±4.41) by fecal antigen ELISA in 600 fecal samples. Significant differences (P ≤ 0.05) in prevalence were found between the three populations. Twenty (10.1%, 95% CI = ±7.86) and 11 (5.6%, 95% CI = ±7.70) of 197 fecal samples from household dogs without clinical signs were positive by microscopy and by antigen ELISA, respectively. Twenty-nine (23.8%, 95% CI = ±6.58) and 23 (18.8%, 95% CI = ±7.81) of 122 the fecal samples from household dogs with clinical signs were positive by microscopy and by antigen ELISA, respectively. Forty-five (16.01%, 95% CI = ±5.62) and 32 (11.3%, 95% CI = ±6.38) of 281 fecal samples from stray dogs were positive by microscopy and by fecal antigen ELISA, respectively. Dogs from the youngest age group (6 months to 1 year) were more likely to be E. histolytica antigen positive than were dogs from the other two older age groups, with a significant difference (P ≤ 0.05) between all age groups. Statistically, no significant (P ≥ 0.05) difference of prevalence was seen in male and female dogs. The local dogs had the highest prevalence rate of E. histolytica antigens (36 of 246, 14.2%, 95% CI = ±6.32) followed by imported breeds (11 of 115, 9.5%, 95% CI = ±10.4) and crossbred (19 of 239, 8.3%, 95% CI = ±7.47), indicating a significant (P ≤ 0.05) trend of positivity between various breeds
∗ Corresponding author. E-mail address: [email protected] (M.M. Nazir). http://dx.doi.org/10.1016/j.vetpar.2014.12.001 0304-4017/© 2014 Elsevier B.V. All rights reserved.
M.A. Alam et al. / Veterinary Parasitology 207 (2015) 216–219
217
of dogs. These findings suggest that dogs may play an important role in the epidemiology of this pathogen. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Amebic dysentery is a disease of man and animals caused by the intestinal protozoan parasite Entamoeba histolytica. It is characterized by severe to moderate gastroenteritis. In advance cases, infection may lead to anemia and liver abscess that can spread to other organs particularly lungs and brain (Roberts et al., 1973; Haque et al., 2003). Globally, amebiasis is the third most prevalent cause of human mortality after malaria and schistomiasis. It is responsible for 40,000 deaths per annum (Sebastiaan et al., 2007). It is estimated that 10% of the world’s population are effected with amoebe predominantly in developing countries in tropical and subtropical areas (Ravdin, 2000; Stanly, 2003). The major source of transmission is untreated drinking water and food contaminated by E. histolytica cysts (Bruckner, 1992). The genus Entamoeba consists of many species but E. histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanni are commonly found in the intestinal lumen of humans and other mammals (Fotedar et al., 2007). E. histolytica was once considered to be a single species, but advance molecular techniques and isoenzyme studies indicated that it be reclassified into two morphologically identical species: the pathogenic E. histolytica and the non-pathogenic E. dispar that are morphologically identical but genetically and biochemically different (Stark et al., 2007). The diagnosis of Entamoeba infection initially relies on fecal examination but due to morphological resemblance of these Entamoeba species it cannot be made by routine staining methods. Pathogenic E. histolytica from identical non-pathogenic E. dispar and E. moshkovskii can only be differentiated by advance methods such as Entamoeba antigen ELISA, DNA detection, PCR and isoenzymes analysis (Markell et al., 1999; Tanyuksel and Petri, 2003; Ximénez and Nosaki, 2014). Dogs are important in the developed and developing countries. Their role as companion and service animals affords them an important position in the human world compared to other domestic animals (Jane, 1996). Because of this association the role of dogs as sources of parasites infective for their human owners is important (Lorenzini et al., 2007). Because of the importance to human health and limited current information on prevalence, we conducted the current study to determine the prevalence of Entamoeba infection in dogs in an endemic area of Lahore, Pakistan.
7,566,000 individuals. The study group was designed by clustered sampling and size of the sample was calculated by online survey system software (sample size calculator). Three dogs populations of different breeds and age groups were evaluated to detect E. histolytica/dispar/moskovskii cysts using light microscopy and for fecal antigens of E. histolytica. A total of 600 fecal samples were obtained from three groups of dogs, the first group consisted of 197 household dogs without clinical signs, group 2 contained 122 household dogs exhibiting clinical signs of diarrhea, dysentery and vomiting were brought to 9 veterinary clinics at various locations in Lahore, Pakistan. Group 3 consisted of 281 stray dogs collected from the city. The study was approved by the Directorate of advance Studies and Research Board, UVAS and Department of Epidemics and Public Health, Lahore. Fecal samples were obtained from the rectum of dogs by using disposable gloves and placed into clean and dry universal glass bottles and taken to the Diagnostic Parasitology Laboratory, Department of Parasitology, University of Veterinary and Animal Sciences, Lahore for further processing. Data regarding epidemiological information on dogs age, sex and breed were obtained from owners, practitioners and researchers at the time of sampling. The date of sample collection was also recorded and used to evaluate the influence on season of the year with parasite prevalence. Information about clinical symptoms i.e. dogs with diarrhea, dysentery, fever and vomiting followed by weight loss in some cases were also recorded on a separate data sheet at the time of sampling. 2.2. Laboratory diagnosis
2. Materials and methods
About 5 g of fecal sample was fixed in methanol and examined by the triple fecal test (Van Gool et al., 2003) to detect fecal cysts with morphology consistent with E. histolytica (referred to as E. histolytica/dispar/moskovskii). A small portion of fecal sample was not fixed but kept at 4 ◦ C until examined for fecal antigens of E. histolytica using a commercially available fecal antigen ELISA kit (Tech Lab, Blacksburg, VA, USA). The samples were processed according to the manufacturer’s instructions. Positive and negative controls were provided with the kit. Because cyst morphology cannot be used to definitively identify E. histolytica, we refer to samples positive for cysts by the triple test as being E. histolytica/dispar/moskovskii. Only samples positive by antigen ELISA were declared to be true E. histolytica positives (Table 1). Triple fecal test positive samples were stained with permanent stain (chlorazole black stain). Samples evaluated by using fecal antigen ELISA were read using an ELISA plate reader (Thermolectron, Finland) operating at 450 nm wavelength.
2.1. Dog populations and sample collection
2.3. Dogs breed and sex
Lahore is the second largest metropolitan city in Pakistan with an area of 1172 km2 and population of
The dogs were classified into exotic breeds, local breeds and crossbreeds (cross between local and exotic breeds).
218
M.A. Alam et al. / Veterinary Parasitology 207 (2015) 216–219
Table 1 Prevalence of Entamoeba histolytica/dispar/moskovskii cysts and E. histolytica antigen positive samples among dog populations. Characteristics
No. examined (N)
Category
Triple fecal test
ELISA
Positive (n)
% (95% CI)
P value
Positive (n)
% (95% CI)
0.011
11
5.58 (±7.70)
23
18.85 (±7.81)
P value
Household dogs without clinical signs Household dogs with clinical signs Stray dogs
197
20
10.15 (±7.86)
122
29
23.77 (±6.58)
281
45
16.01 (5.62)
32
11.38 (±6.38)
Sex
Male Female
362 238
58 36
16.0 (±4.95) 15.12 (±6.26)
0.768
45 21
12.4 (±5.47) 8.8 (±7.40)
0.167
Age group
6 months to 1 year 1–6 year >6 year
145 170 285
36 28 30
24.82 (±5.79) 16.47 (±7.12) 10.52 (±6.48)
0.001
27 19 20
18.6 (±7.22) 11.2 (±8.24) 7.0 (±7.06)
0.0012
Breed
Exotic Local Crossbred
115 246 239
16 49 29
13.9 (±9.33) 19.9 (±5.31) 12.1 (±6.79)
0.000
11 36 19
9.5 (±10.4) 14.2 (±6.32) 8.3 (±7.47)
0.001
Total
600
94
15.66 (±3.88)
66
11.0(±4.41)
Dogs group
Dog breed was defined based on pedigree standards of the Kennel Club of Pakistan. A dog was placed in the crossbred group if it possessed the phenotype of two distinct breeds and all native dog breeds were compiled in local breeds. Each dog’s sex was recorded as male or female despite of if the animal had been neutered or spayed and the dogs were classified into 3 different age groups (Table 1). Age groups were 6 months to 1 year (N = 145), 1–6 years (N = 170), greater than 6 years (N = 285). 2.4. Statistical examination The percentage estimates 95% confidence level was calculated by Microsoft Excel 2010 software and the values between different populations are compared by Pearson’s x2 test with respect to age, breed and sex using (SPSS for Windows, version 20.0; SPSS, Chicago, IL), and P ≤ 0.05 was considered as significant prior to conduct of the test. 3. Results The results showed that out of 600 fecal samples examined 94 (15.6%, 95% CI = ±3.88) were positive for E. histolytica/dispar/moskovskii cysts by microscopy while 66 (11%, 95% CI = ±4.41) of samples were found positive by antigen ELISA (Table 1). Prevalence rates of E. histolytica/dispar/moskovskii cysts (29/122, 23.77%, 95% CI = ±6.58) and E. histolytica antigens (23/122, 18.85%, 95% CI = ±7.81) were highest in household dogs with clinical signs, stray dogs were the next group having high prevalence of E. histolytica/dispar/moskovskii cysts (45/281, 16.01%, 95% CI = ±5.62) and E. histolytica antigens (32 of 281, 11.38%, 95% CI = ±6.38), whereas the household dogs without clinical signs had the lowest prevalence of E. histolytica/dispar/moskovskii cysts (20/197, 10.15%, 95% CI = ±7.86) and E. histolytica antigens (11 of 197, 5.58%, 95% CI = ±7.70). The prevalence rates were significantly (P ≤ 0.05) different within three populations of dogs both by microscopy and antigen ELISA (Table 1). Likewise
0.012
prevalence rates were significantly (P ≤ 0.05) different between dogs with and without clinical signs (Table 1). Our results demonstrated that 17 of 94 (18%) samples positive by light microscopy were negative by fecal antigen ELISA. Five (17.2%) samples from household dogs with clinical signs, three (15%) from household dogs without clinical signs and 9 (19.4%) from stray dogs were triple fecal test positive samples that were negative for fecal antigen ELISA. These findings represent the potential prevalence of E. dispar and E. moshkovskii in dog’s population. Male dogs were more likely to be positive for E. histolytica/dispar/moskovskii cysts (58/362, 16%, 95% CI = ±4.95) and E. histolytica antigens (45 of 362, 12.4%, 95% CI = ±5.47), than female dogs which had a prevalence of E. histolytica/dispar/moskovskii cysts (36/238, 15.12%, 95% CI = ±6.26) and E. histolytica antigens (21 of 238, 8.8%, 95% CI = ±7.40). This was not statistically (P≥ 0.05) significant (Table 1). The prevalence of dogs that were positive for E. histolytica/dispar/moskovskii and for E. histolytica antigens by ELISA was highest in the youngest age group (6 months to 1 year) (Table 1). Prevalence of infection based on age group was significantly (P ≤ 0.05) different using either triple fecal test or E. histolytica antigen ELISA. The prevalence of E. histolytica/dispar/moskovskii cysts and E. histolytica fecal antigen positive samples was interlinked with season of the year. Most of the positive cases were observed during the summer season and fewest in the winter months. The local dogs had the highest prevalence of E. histolytica/dispar/moskovskii cysts (49/246, 19.9%, 95% CI = ±5.31) and E. histolytica antigen (36 of 249, 14.2%, 95% CI = ±6.32) followed by exotic and crossbred (Table 1). However, no significant (P ≤ 0.05) difference of prevalence among different dog breeds was observed using both microscopy and antigen ELISA. 4. Discussion Amoebic dysentery, an intestinal disease caused by infection with the protozoan parasite E. histolytica, is an
M.A. Alam et al. / Veterinary Parasitology 207 (2015) 216–219
important disease of man and animals throughout many areas of the world. In the present study a high prevalence of E. histolytica/dispar/moskovskii cysts were recorded in dogs by triple fecal test and relatively low prevalence rate of true E. histolytica infection was observed in dogs based on fecal antigen ELISA. However, the difference of prevalence recorded by two tests was statistically significant (P ≤ 0.05). As triple fecal test cannot differentiate between potentially pathogenic E. histolytica and non-pathogenic but morphologically indistinguishable E. dispar or E. moskovski so all cysts viewed by light microscopy were called E. histolytica/dispar/moskovskii. The fecal antigen ELISA is specific for E. histolytica and does not cross react with E. dispar and is considered as true E. histolytica infection in the current report. The prevalence rate is analogous to the formerly reported study based on microscopy with high prevalence rate in dogs (Johnson et al., 2010). The current findings of E. histolytica antigen positive dog samples gives us a clue that dogs can play an important role in the epidemiology of Entamoeba infection in endemic areas. The highest prevalence rate of infection was observed in symptomatic household dogs as compared to stray dogs and asymptomatic household dogs (Table 1). The higher prevalence of Entamoeba infection in household symptomatic dogs may be due to close association of dogs and humans. Ingestion of contaminated food and water may be a possible source of transmission of infection between dogs and humans. The sex wise prevalence of this disease was observed as, male dogs had higher prevalence than the female dogs. However, in an earlier study, female dogs had higher prevalence than male dogs (Mahfooz et al., 2014). Our results confirmed that the prevalence of infection with E. histolytica/dispar/moskovskii cysts and E. histolytica fecal antigens decreased with the dog’s age (Table 1) and it was highest in the youngest age group as previously, reported by others (Sarger et al., 2006b, Lorenzini et al., 2007; Martinez-Moreno et al., 2007). The highest prevalence rate of infection in young dogs might be due to immuno incompetence as a result of reduced level of passive immunity received from the mothers (OliveriaSequeira et al., 2002). We found a significant (P ≤ 0.05) difference in prevalence of Entamoeba infection among different breeds of dogs and in local dogs compared to other groups of dogs. This corresponds to previous observations made by Johnson et al. (2010) and Mahfooz et al. (2014). These reports should be interpreted carefully because housing conditions may influence transmission of Entamoeba species in dogs.
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Acknowledgments The current study was supported by a scholarship to M.A.A. from the Higher Education Commission, Islamabad, Pakistan. The authors would like to thank Dr. Atta Subhani, Muhammad Asif Ali, Dr. M. Oneeb, Dr Tanveer ul Haq, Dr. Ahsan Mustafa, Noman Nazir, and Muhammad Kashif Nazir for their moral enthusiasm and technical assistance during this study. References Bruckner, D.A., 1992. Amebiasis. Clin. Microbiol. Rev. 5, 356–369. Fotedar, R., Stark, D., Beebe, N., Marriott, D., Ellis, J., Harkness, J., 2007. PCR detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from Sydney, Australia. J. Clin. Microbiol. 45, 1035–1037. Haque, R., Huston, C.D., Hughes, M., Houpt, E., Petri, W.A., 2003. Amebiasis. N. Engl. J. Med. 348, 1565–1573. Jane, E., 1996. Collins Gem Dogs Photoguide, first ed., pp. 5–6. Johnson, O., Adejinmi, Joseph, O., 2010. Prevalence of intestinal protozoan parasites of dogs in Ibadan, south western Nigeria. J. Anim. Plant Sci. 7, 783–788. Lorenzini, G., Tasca, T., De Carli, G.A., 2007. Prevalence of intestinal parasites in dogs and cats under veterinary care in Porto Ategre, Rio Grande do Sul, Brazil. Braz. J. Vet. Res. Anim. Sci. 44, 137–145. Mahfooz, A., lqbal, M., Ahmad, T., Khan, M., Iqbal, A., Saleem, I., 2014. Amoebiasis: prevalence, treatment with metronidazole and nalidixic acid in dogs. Sch. Ad. Anim. Vet. Res. 1, 13–15. Markell, E.K., John, D.T., Krotoski, W.A., 1999. Medical Parasitology, eighth ed. Saunders Company, Mexico, pp. 24–89. Martinez-Moreno, F.J., Hernandez, S., Lopez, C.E., Becerra, C., Acosta, I., Martinez Mareio, A., 2007. Estimation of canine intestinal parasites in Cordoba (Spain) and their risk to public health. Vet. Parasitol. 143, 7–13. Oliveria-Sequeira, T.G., Amarante, A., Ferrari, T.B., Nunes, L.C., 2002. Prevalence of intestinal parasites in dogs from Sao Panto State, Brazil. Vet. Parasitol. 103, 19–27. Ravdin, J.I., 2000. Amoebiasis. Imperial College Press, London, pp. 47–66. Roberts, E.D., Williams, J.C., Piie, G., 1973. Naturally occurring gastric amebiasis in the wallaroo. Vet. Pathol. 10, 323–329. Sarger, H., Steiner, M.C., Multer, N., Staubli, D., Esposito, M., Schares, G., Hassig, M., Stark, K., Gottsterin, B., 2006b. Incidence of Neospora caninum and other intestinal protozoan parasites in populations of Swiss dogs. Vet. Parasitol. 139, 84–92. Sebastiaan, J., Van Hal, J.V., Stark, D.J., Fotedar, R., Marriott, D., John, T., Ellis, J.T., Harkness, J.L., 2007. Amoebiasis: current status in Australia. Med. J. Aust. 186, 412–416. Stanly, S.L., 2003. Amoebiasis. Lancet 361, 1025–1034. Stark, D., Fotedar, R., Van Hal, S., Beebe, N., Marriott, D., Ellis, J.T., Harkness, J., 2007. Prevalence of enteric protozoa in human immunodeficiency virus (HIV)-positive and HIV-negative men who have sex with men from Sydney. Aust. Am. J. Trop. Med. Hyg. 76, 49–552. Tanyuksel, M., Petri, W.A., 2003. Laboratory diagnosis of amoebiasis. Clin. Microbiol. Rev. 16, 713–729. Van Gool, T., Weijts, R., Lommerse, E., Mank, T.G., 2003. Triple faeces test: an effective tool for detection of intestinal parasites in routine clinical practice. Eur. J. Clin. Microbiol. Inf. Dis. 22, 284–290. Ximénez, C., Nosaki, T., 2014. Metabolomic analysis of Entamoeba: applications and implications. Curr. Opin. Microbiol. 20, 118–124.