Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq
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MOLECULAR DETECTION OF Toxoplasma gondii IN CHICKEN
LICE (Menacanthus stramineus)
Sura E. Ahmed* Nadia K. Thamar* Rasha M. Othman *
*Department of Veterinary Microbiology and Parasitology, College of Veterinary
Medicine, University of Basrah, Basrah, Iraq.
Corresponding Author: rashamunther2014@yahoo.com
Keywords: ectoparasites, Toxoplasma gondii, chickens.
ABSTRACT
The current study was carried out to demonstrate the ability of chicken lice
Menacanthus stramineus to transmit Toxoplasma gondii by using PCR technique. The
blood was collected from the jugular vein of 85 chickens infested with ectoparasites
to investigate the existence of Toxoplasma gondii by latex agglutination test. The
results showed that 42 (49.41%) of chicken were infected with lice, lice samples of
Menacanthus stramineus were collected from chickens infected with T.gondii for
PCR investigation to confirm the presence of T.gondii in lice tissues and the results
were revealed the presence of T. gondii B1 genein 18(42.85 %) samples.
INTRODUCTION
Poultry is a domesticated birds kept for the production of meat and eggs. This
involves birds like chicken, goose, turkey, duck, pheasant, quail, guinea fowl and
peafowl. Chickens are the most important poultry worldwide irrespective of
civilization and religion because chicken products have very high nutritive values.
Chickens are an economic and efficient supply of animal protein within the shortest
possible time, playing a vital role in narrowing down the animal protein source gap
(1,2). Like all other animals, backyard poultry too suffer from a wide range of
diseases. In semi-intensive system, chickens are found to be infested with several
species of ectoparasites including different species of lice, mites etc (3,4). Lice
consider one of the biggest entirely parasitic insect order and their all members are
obligate ectoparasites of mammals and birds. According to the last researches
approximately 4500 which described louse species. Moreover, the majority of these
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq
17
species (more than 85%) are infests birds. Based on classification, they have its
place to the Exopterygota cluster of insects and are classified into 4 sub orders which
including: Amblycera, Ischnocera, Rhynchophthirina, and Anoplura. The first
divisions as Mallophaga (chewing lice) and Anoplura (sucking lice) have been no
longer to be uses anymore such as Mallophaga turned out to be paraphyletic (5).
The first hugest problems for lice is the mode of transition to a new host individual,
as they are wingless, the spreading usually includes direct physical contact
between host individuals (6). Toxoplasmosis is a zoonotic disease broadly distributed
through and all over the globe. It is caused by the intracellular blood parasite called
Toxoplasma gondii. On the other hand, the domestic cats and the entire cat family are
consider the main host of the parasite, where in the parasite reproduces sexually. T.
gondii can also infect a varied types of intermediate hosts, the most affecting are the
warm-blooded animals (7). T. gondii is a parasite that is often distribution through the
world, with a higher incidence in tropical regions and a decrease when the latitude
upsurges (8). The felids play significant role in the spreading of T. gondii to humans
and other animals, through excretion of environmentally resistant oocysts in their fece
(9). Moreover, the characterization and identification of T. gondii has been done in
several animal species including wild, companion and production animals (10). The
first protocol for molecular detection of T.gondii, for standard PCR targeting B1
sequence, was developed in 1989 and has since been changed and optimized in
several laboratories (11, 12). The B1 sequence, though of unknown perform, is wide
exploited in a variety of diagnostic and epidemiologic studies due to its specificity
and sensitivity (13) .The present study was conduct to prove the role of chewing lice
in transmission of Toxoplasma gondi to chicken by polymerase chain reaction (PCR ).
MATERIALS AND METHODS
A total of 85 Chickens infested with ectoparasites were collected from
different areas in Basrah Governorate. Blood sample was withdrawn from the jugular
vein (5 ml) of the infected chicken and placed in a vacuum tube (not contain
anticoagulant). Then, the tubes was centrifuged at 3,000 rpm for 5 min to collect
serum to investigate the presence of T.gondii using latex agglutination test.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq
18
Latex agglutination assay: Detection of Toxoplasma gondii in serum of the infected
chicken was done using latex agglutination kit (Netherland, Saluce). Briefly, Forty
microliter of the serum were distributed on the single circle of the test slide, this step
was repeated for the positive and negative controls. Serum was then distributed over
the entire test circle. The toxo latex reagent was shaken well and the reagent suck up
with the needle of the kit. After that, one fall drop of the latex reagent was distributed
on each test circle that containing sample. Finally, the mixture was mixed well for 4
min. The agglutination was then examined under 4x force of dissecting microscope.
Polymerase Chain Reaction (PCR) assay: Detection B1 gene of Toxoplasma
gondii in lice tissue was done using PCR technique. The lice sample was prepared
for DNA extraction by crushed twenty five milligrams of lice (Menacanthus
stramineus) by using the mortar and pestle. The sample was then transferred in the
eppendroff tubes (1.5 ml). The DNA extracted steps from the lice tissue was done
according to the procedure of the AccuPrep ® Genomic DNA Extraction kit (Bioneer,
Korea).
The PCR technique used to detect the T. gondii B1 gene using specific
foreword primer (5-GAACCACCAAAAATCGGAGA-3) and reverse primer (5-
GATCCTTTTGCACGGTTGTT-3), which amplify (399bp) verified as positive for
B1gene (14).The reaction of PCR was consisted of Master Mix(3 μl), forward primer
(0.5μl), reverse primer (0.5μl), and DNA templates (10μl). The final volumes of the
reaction were 20 μl. by adding nuclease-free water (6 μl ) The PCR amplification
method was accomplished by employing a Thermocycler (Esco, Singapore), with
athletics conditions consisting of initial denaturation at 94°C for five min, followed
by thirty cycles of amplification (denaturation at 94°C for thirty sec, tempering at
53°C for thirty seconds, extension at 72°C for forty five sec, and final elongation at
72°C for five min). The PCR products were analyzed using 2% of agarose gel
electrophoresis. Molecular weight marker a 100 bp of DNA ladder (Bioneer, Korea)
was used. The gel was stained with ethidium bromide and visualized under ultraviolet
(UV) illumination (E-graph – ATTO/Japan).
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding o
College of Veterinary Medicine. University of Basrah. Iraq
A total of 85 sera of chickens infested with ectoparasites were tested by latex
agglutination test for detection of
were positive and the rate of infection was 49.41% .
isolates from lice tissue were conducted for PCR assay to detect the presence of
gene and the results of PCR amplification of
revealed that 18 (42.85 %) isolates had clear band of approximately 399bp which
corresponded to identification of
Figure 1 :PCR amplification results for
Ladder. Lane 2:negative control. Lanes 3
(399bp), lane 7 negative PCR result.
Ectoparasites are frequently found in all bi
blood, feathers, skin, and scales. T
including discomfort, irritation, loss of feathers, growth was stunted and the eggs
production and hatchability were
mortality, and susceptibility to different infections
The result of latex Agglutination test for
result was closed to result that obtained by (
of the 17th International
19
RESULTS
T.gondii. The current study showed that 42 samples
DNA from 42 samples
B1gene of T.gondii from lice tissue
T.gondii (Figure 1).
B1 gene of T. gondii. M: 100 bp of DNA
ve 3-6: positive PCR result for
DISCUSSION
birds, where they nourish
This is may cause a series of a symptoms, which
reduced egg, anemia, increased feed costs, elevated
(15).
lt T.gondii was 49.41 % (42/85). This
16) in Austria and (17)
f Conference.
B1
B1 gene
rds, on their
in Iran as they
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq
20
reported seroprevalence of 40% and 43% respectively in chicken. On the other hand,
(18) found that the high seroprevalence of T. gondii in chicken is 9%.While, (19)
reported 81% in Nineveh in Broiler chicken. The management and hygienic standards
in breeding, density of felines and different environmental conditions are effective
factors on the gaining of T .gondii oocysts by animals (20). The rate of infection in
free-ranging chicken is a crucial indicator of environmental contamination as a result
of food habits (21, 22).
PCR technique was applied in the present study to detect the pathogenic
protozoan T.gondii in tissue of lice (Menacanthus stramineus) and to confirm the
result of latex agglutination test. Result of PCR technique for detection of B1 gene
(399bp) in 42 samples revealed only 18 samples were positive with a rate 42.85 %.
This may confirms the ability of lice to transfer T.gondii from infested chicken to
healthy chicken as a result of transmission between chicken and feed on their blood.
It has been found that Menacanthus stramineus have the ability to transmit T.gondii
(23). In addition, the ability of lice to transport the T.gondii among chicken leads to
an increase in cases of infection with T.gondii (24,18). Previously, detection of
T.gondii in lice tissue has been detected using B1 gene with percentage 62.8 % in al-
AL-Diwaniyah Governorate, Iraq (25).
B1 gene has a high specificity in T. gondii and has been repeated 35 times in
its genome, so it is used as a target for amplification in polymerase chain reaction to
detect parasites in clinical materials such as the blood and tissues (11). Additionally,
the number and concentration of such organisms in a such sample play critical point
in concentration of DNA that extract with the influence of the types and kit procedure
used for purified the T.gondii from the ectoparasites. All these factors might be the
reason for low percentage of infection in the present study.
REFERENCES
1-Al-Nasser, A., Al-Khalaifa, H. , Al-Saffar, A., Khalil, F., Albahouh, M.,
Ragheb, G., and Mashaly, M. (2007). Overview of chicken taxonomy
and domestication. World's Poultry Science Journal, 63(2): 285-300.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq
21
2-Nadeem, M., Khan, M. N., Iqbal, Z., Sajid, M. S., Arshad, M., and Yaseen, M.
(2007). Determinants influencing prevalence of louse infestations on layers
of district Faisalabad (Pakistan). British Poultry Science, 48(5): 546-550
3-Zeryehun, T., and Fekadu, G. (2012). Seroprevalence of infectious bursal disease
in chickens managed under backyard production system in Central Oromia,
Ethiopia. African Journal of Microbiology Research, 6(38), 6736-6741.
4-Meguini, M. N., Righi, S., Zeroual, F., Saidani, K., and Benakhla, A. (2018).
Inventory of lice of mammals and farmyard chickens in North-Eastern
Algeria. Veterinary World, 11(3): 386
5-Vas, Z. (2014). Host-parasite relationship of birds (Aves) and lice (Phthiraptera)–
evolution, ecology and faunistics .PhD thesis, School of Veterinary
Science. Budapest, Hungary .
6-Durden, L. A. (2019). Lice (Phthiraptera). In Medical and Veterinary
Entomology. Academic Press: 79-106
7-Amouei, A., Sarvi, S., Sharif, M., Aghayan, S. A., Javidnia, J., Mizani, A., ... &
Nayeri Chegeni, T. (2020). A systematic review of Toxoplasma gondii
genotypes and feline: Geographical distribution trends. Transboundary and
emerging diseases, 67(1), 46-64.
8-Wang, L., Chen, H., Liu, D., Huo, X., Gao, J., Song, X., ... and Lu, F. (2013).
Genotypes and mouse virulence of Toxoplasma gondii isolates from
animals and humans in China. Plos one, 8(1): e53483.
9-Yan, C., Liang, L. J., Zheng, K. Y., and Zhu, X. Q. (2016). Impact of
environmental factors on the emergence, transmission and distribution of
Toxoplasma gondii. Parasites and Vectors, 9(1): 137.
10Cenci-Goga, B. T., Rossitto, P. V., Sechi, P., McCrindle, C. M. and Cullor, J. S.
(2011). Toxoplasma in animals, food, and humans: an old parasite of new
concern. Foodborne Pathogens and Disease, 8 (7): 751-762.
11-Reischl, U., Bretagne, S., Krüger, D., Ernault, P., and Costa, J. M. (2003).
Comparison of two DNA targets for the diagnosis of Toxoplasmosis by
real-time PCR using fluorescence resonance energy transfer hybridization
probes. BMC Infectious Diseases, 3(1): 7.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq
22
12-Switaj, K., Master, A., Skrzypczak, M., and Zaborowski, P. (2005). Recent
trends in molecular diagnostics for Toxoplasma gondii infections. Clinical
Microbiology and Infection, 11(3): 170-176.
13-Al-Sray, A. H., Sarhan, S. R. and Mohammed, H. A. (2019). Molecular and
serological characterization of Toxoplasma gondii in women in Wasit
province. Advances in Animal and Veterinary Sciences, 7(8): 657-663.
14-Al-Lebawi F. I. and AL-Mayali H. M. (2015). Molecular detection of
Toxoplasmosis in biting lice (Menacanthus stramineus) on naturally
infected chickens using polymerase chain reaction. Al-QadisiyahJouranl of
Veterinary Medicine Sciences, 14 (2): 37-40.
15-Ola-Fadunsin, S. D., Gimba, F. I., Abdullah, D. A., and Konto, M. (2018).
Epidemiology of Avian Ectoparasitic Infestations Diagnosed in Osun State
Veterinary Hospitals, Southwest Nigeria. Notulae Scientia
Biologicae, 10(2), 143-147.
16-Dubey, J. P., Navarro, I. T., Graham, D. H., Dahl, E., Freire, R. L.,
Prudencio, L. B., and Lehmann, T. (2003). Characterization of
Toxoplasma gondii isolates from free range chickens from Paraná,
Brazil. Veterinary Parasitology, 117(3): 229-234.
17-Zia-Ali, N., Keshavarz-Valian, H., Rezaian, M., Khorramizadeh, M. R.,
Kazemi, B., Fazaeli, A., and Darde, M. (2005). Molecular
characterization of Toxoplasma gondii from bird hosts. Iranian Journal of
Public Health: 27-30.
18-Zakaria, E. G. (2011). Detection of Toxoplasma gondii antibodies in different
meat juices. Rafidain journal of Science, 22(4E): 17-25.
19-Mahmood, A. F.; Nashwan, A. A.; Waked, H. M.; Bashar, M .J. and Yaser,
Y.H. (2006). Detection of Toxoplasma gondii antibodies in Broiler
chickens in Ninevah governorate. Journal of Dohuk University, 9:145-148.
20-Jones, J. L., Kruszon-Moran, D., Sanders-Lewis, K. and Wilson, M. (2007).
Toxoplasma gondii infection in the United States, 1999–2004, decline from
the prior decade. The American Journal of Tropical Medicine and Hygiene,
77(3): 405-410.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
College of Veterinary Medicine. University of Basrah. Iraq
23
21-Araujo, F. G., and Remington, J. S. (1975). IgG antibody suppression of the
IgM antibody response to Toxoplasma gondii in newborn rabbits. The
Journal of Immunology, 115(2): 335-338.
22-Devada, K., Anandan, R., and Dubey, J. P. (1998). Serologic prevalence of
Toxoplasma gondii in chickens in Madras, India. The Journal of
Parasitology, 84(3): 621-622.
23-Saxena, A. K.; Agarwal, GP.; Chandra, S. and Singh, O.P. (1985). Patho -
genic involvement of Mallophaga .Zur Angewandte Entomologie, 99: 294-
300 .
24-Al-Jubouri, S. A. A. (2010). Endo and ectoparasitic infections in domestic fowl
Gallus gallus domestcus (Linnaeus, 1758). In: Al-Diwaniya city. M.Sc.
Thesis, College of Education, University of Al- Qadisiya, Iraq. (In arabic ).
25-AL-Shaibani, K. T. M. , Al-abodi, H. R. J. and Mahmood, H. R. (2018). Effect
of Two Species of Ticks and Lice on Some Blood and Biochemical
Parameters and their Role in the Transmission of Toxoplasma Gondii to
Turkey Meleagris Gallopavo during Spring and Summer Seasons in ALDiwaniyah
Province – Iraq. Journal of Global Pharma Technology,
10(06):273-280 .
