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Abanico veterinario
On-line version ISSN 2448-6132Print version ISSN 2007-428X
Abanico vet vol.11 Tepic Jan./Dec. 2021 Epub Nov 08, 2021
https://doi.org/10.21929/abavet2021.29
Original papers
Molecular detection of Ehrlichia canis and Anaplasma phagocytophilum and hematological changes of infected dogs
1
http://orcid.org/0000-0003-1282-8713
1
http://orcid.org/0000-0003-4261-5655
1
http://orcid.org/0000-0002-6820-2974
1Facultad de Medicina Veterinaria y Zootecnia “Dr. Norberto Treviño Zapata”. Universidad Autónoma de Tamaulipas, km 5, Carretera Victoria-Mante, Ciudad Victoria, Tamaulipas, CP 87000, México.
Ehrlichiosis and anaplasmosis are tick-borne diseases caused by bacteria of the genera Ehrlichia and Anaplasma. Since clinical manifestations are varied and nonspecific, the diagnosis in clinical practice, remains a challenge for veterinarians. Furthermore, the distribution of these infections includes areas where its tick vector, Rhipicephalus sanguineus is present. This study was designed to evaluate the prevalence and factors associated with the presence of Ehrlichia canis and Anaplasma phagocytophilum in dogs from the central area of Tamaulipas. PCR screened 384 canine blood samples obtained from different veterinary clinics and a shelter. The data were analyzed using the Chi-square test (P level <0.05 for statistical significance). The results showed that 103 (26.8%) out of 384 samples were positive for E. canis, while A. phagocytophilum was not detected. Statistical analysis did not show relationship between E. canis and variables like gender, breed, and origin (P˃0.05). Nonetheless, there was a statistically significant difference between infected adult dogs (15-84 months) compared to other age groups evaluated (p<0.05). Regarding hematocrit, platelets count, plasma protein, total and differential white blood cells counts, none of these parameters were significantly different (P>0.05).
Keywords: Ehrlichia canis; Anaplasma phagocytophilum; PCR; hematological findings
Las ehrlichiosis y anaplasmosis canina son enfermedades transmitidas por garrapatas, provocadas por bacterias del género Ehrlichia y Anaplasma. Debido a sus múltiples manifestaciones clínicas, su diagnóstico es un reto para el veterinario. La distribución de estos hemoparásitos incluye áreas donde su principal vector, Rhipicephalus sanguineus está presente. Este estudio fue diseñado para determinar la presencia Ehrlichia canis y Anaplasma phagocytophilum, así como los factores asociados y hallazgos hematológicos comunes en perros de la zona centro de Tamaulipas. Se evaluaron, a través de PCR, 384 muestras de sangre provenientes de animales de diferentes clínicas veterinarias y un refugio. El análisis de datos se realizó con la prueba Chi cuadrada con un nivel de significancia de 0.05. Los resultados muestran que, del total de muestras 103 (26.8%) resultaron positivas a E. canis, mientras que para A. phagocytophilum no se detectó ningún caso. No se observó asociación significativa con relación al sexo, raza, ni lugar de procedencia (p>0.05), a diferencia de la edad, donde se encontró mayor prevalencia de E. canis para adultos (15-84 meses) (p<0.05). En relación con el hematocrito, conteo de plaquetas, proteínas plasmáticas totales, conteo y diferencial leucocitario, no existió diferencias significativas (p>0.05).
Palabras claves: Ehrlichia canis; Anaplasma phagocytophilum; PCR; valores hematológicos
INTRODUCTION
Ehrlichiosis and anaplasmosis are diseases of great importance for both veterinary and public health, as they are responsible for diseases such as Monocytic Ehrlichiosis and Human Granulocytic Anaplasmosis (Vieira et al., 2013; Farhan 2015; Rodríguez-Vivas et al., 2019). Gram-negative obligate intracellular bacteria cause them. They are known as Ehrlichia spp and Anaplasma spp respectively (Harrus and Waner 2011; Stuen et al., 2013). Worldwide, cases have increased considerably in recent years mainly in tropical and subtropical areas where tick vectors (Ripicephalus sanguineus and Ixodes spp) proliferate (Beugnet and Chalvet-Monfray 2013;Irwin 2014;Little et al., 2014;Battilani et al., 2017). Due to the increasing proximity of people to their pets, the likelihood of bites by these ectoparasites is increasing considerably leading to these infections becoming reemerging zoonoses (Bhadesiya and Modi 2015;Ismail and McBride 2017).
In Mexico, canine Ehrlichiosis was reported for the first time in 1996, since then the number of cases has increased considerably (Maggi and Krämer 2019); however, the diagnosis, in many occasions is based on clinical signs without performing laboratory tests that directly or indirectly corroborate its presence. Definitive diagnosis focuses on microscopic techniques; however, these methods have low sensitivity and specificity in patients with low bacteremia, which prevents establishing adequate therapeutics (Harrus and Waner 2011;Allison and Little 2013). In response to this, Polymerase Chain Reaction (PCR) emerges as an important tool to support conventional diagnostic methods (Almazan et al., 2016;Cetinkaya et al., 2016;de la Fuente et al., 2017).
Tamaulipas state due to its geographical location has ideal characteristics that favor the development of ticks vectoring these diseases (Tinoco-Gracia et al., 2009); however, the true magnitude of this problem is unknown. Therefore, the main objective of this research was to determine the presence of E. canis and A. phagocytophilum, through PCR, in naturally infected dogs in the central zone of Tamaulipas; as well as to evaluate some factors associated with the presence of these diseases.
MATERIAL AND MÉTODOS
Study area
The present work was carried out with blood samples from dogs submitted (during the period March 2020 to March 2021) to the Laboratory of Parasitology and Clinical Analysis of the Faculty of Veterinary Medicine and Zootechnics "Dr. Norberto Treviño Zapata", belonging to the Autonomous University of Tamaulipas. Also, samples from several private veterinary clinics in the capital of Tamaulipas and some surrounding municipalities.
Study population
A non-probabilistic sampling was used. Samples from patients referred with the following inclusion criteria were analyzed: 1) being from Tamaulipas state (central zone), 2) presenting clinical signs related to hemoparasites (fever, diarrhea, uveitis, petechiae, epistaxis, osteoarticular, and respiratory, reproductive and neurological disorders), 3) presenting or having been in contact with ticks, and 4) having the consent of the pet owner. The sample size was 384 animals, which is the minimum sample size obtained from the formula of (n) for infinite population proportions, since there is no canine population census in the area to be evaluated (Wayne and Chad 2013). All dogs were handled according to the official animal welfare standards established by the Bioethics Committee of the Faculty of Veterinary Medicine and Zootechnics of the Autonomous University of Tamaulipas.
Sample collection
A minimum of 3 ml of blood was obtained by venous puncture (cephalic vein), which were rapidly transferred to a tube (BD Vacutainer®) with EDTA K2 (ethylenediaminetetraacetic acid potassium) anticoagulant. Samples were kept refrigerated (8°C) for no more than 24 hours before processing for hematological evaluation. An aliquot of blood was saved in 1.5 ml vials and was stored at -20°C for subsequent DNA extraction and PCR testing. In all cases, the age, sex, pedigree and season of the year in which the sample was taken from the individuals studied were recorded.
Hematological analysis
The determination of hematological parameters was performed immediately, within 4 hours of blood collection to avoid morphological alterations of cells. The samples were analyzed in an automated equipment (Auto Hematology Analyzer, MINDRAY, BC-2800 Vet; Shenzhen, China). For the determination of plasma proteins, the microhematocrit method was used, using capillary tubes without heparin; which were filled with – parts with blood, sealed and centrifuged (centrifuge KHT-410E Kendal Import S.A.C Gemmy Taiwan) at 11,500 rpm for 5 min. The plasma obtained was placed in a refractometer (American Optical) and total proteins were obtained. The leukocyte differential count was performed manually. The first consisted of assessing and counting in a blood smear (stained with Diff-Quik™) 100 nucleated cells and thus obtaining the percentage count of the different leukocytes: neutrophils, eosinophils, lymphocytes, monocytes and basophils. To determine whether anaemia was present, the hematocrit value was taken into account, which was categorized into 2 groups, with and without the presence of anaemia. The platelet count and total protein were divided into 2 groups, animals with and without thrombocytopenia and with and without the presence of hyperproteinemia, respectively. Total leukocytes as well as their different populations were grouped as normal, high and decreased counts.
Identification of hemoparasites by microscopy
For the search for hemoparasites by microscopy, blood smears were prepared, fixed with methanol for 5 minutes and stained with 10% Giemsa solution for 15 min. Subsequently, multiple random areas of the monolayer and tail of the smear were evaluated under the microscope with the immersion objective (100x); here we looked for the presence of morulae (cytoplasmic aggregates of basophilic color) or any other inclusion body compatible with hemoparasites (Dulmer et al., 2001).
MOLECULAR ANALYSIS
Obtaining nucleic acids
From the stored EDTA blood aliquots, DNA extractions were performed using the commercial DNA extraction and purification kit (Wizard® Genomic DNA Purification- Promega), according to the protocols established by the company. The total DNA extracted was quantified, using a spectrophotometer (NanoDrop2000®, Thermo Scientific, Waltham, MA, USA) and stored at -20 °C until further use in PCR assays.
Polymerase Chain Reaction
For molecular analysis, a region of the GltA gene (used for identification of rickettsiae coding for the enzyme citrate synthase) for E. canis and a region of the Msp4 gene (major surface complex) for A. phagocytophilum were amplified. The GoTaq® Green Master Mix kit (Promega, Madison, WI USA. Cat. Num: M7122) was used according to protocols established by the company. For this, 21 µl of kit solution, 1 µl of sense primer, 1 µl of antisense primer and 2 µl of DNA from each sample were used to reach a final volume of 25 µl. The samples were then amplified in the thermal cycler (Applied Biosystems™ Num: 2720) with the amplification protocol shown in Table 1. The amplified products were analyzed by 2% agarose gel electrophoresis in 600 ml TAE Buffer, 1X (Promega, Madison, WI USA. Cat. Num: V4271) at 120 V for 40 min using the nucleic acid dye Diamond Nucleic Acid Dye (Promega, Madison, WI USA. Cat.Num: H1181) and subsequently visualized under UV light from the UVP transilluminator (Ultraviolet Products, Inc., California, USA. Cat. Num: TFM-30). DNA fragments of known lengths (E. canis, 200 bp; A. phagocytophilum: 980 bp) and a 100 bp DNA Ladder molecular weight marker (Promega, Madison, WI USA. Cat. Num: G210A) were used as a positive control for reference.
Table 1 Sequence of oligonucleotides used for each pathogen, amplification protocol and size of amplified
| Organism and target gene | Oligonucleotide sequence (5'-3') | Amplification program | Amplified size (bp) | Reference |
|---|---|---|---|---|
| E. canis Fw | 95°C for 5 min | |||
| E. canis | ATAAACACGCTGACTTTACTGTTCC | 94°C for 30 s 60 °C for 30 s | 200 | Stich et al., 2002 |
| (GltA) | E canis Rev | 72 °C for 1 min | ||
| GTGATGAGATAGAGCGCAGTACC | 72 °C for 7 min | |||
| 35 cycles | ||||
| A. phagocytophilum | MSP4AP5 ATGAATTACAGAGAATTGCTTGTAGG | 94°C for 5 min 94°C for 30 s 50 °C for 30 s | 849 | Yousefi et al., 2019 |
| (Msp4) | MSP4AP3 TTAATTGAAAGCAAATCTTGCTCCTATG | 72 °C for 30 s 72 °C for 7 min 35 cycles |
Statistical analysis
Absolute frequencies and percentages of positive cases and hematological findings were represented. The degree of association between the presence of pathogens and the variables evaluated (sex, age, pedigree, time of year and hematological parameters) were analyzed by the Chi-square test of independence with a significance level of 0.05, using the statistical program MedCalc. V. 7.0.
RESULTS
Presence of E. canis and A. phagocytophilum
From the total number of samples analyzed (384) during the study period, 103 were positive for E. canis (frequency of 26.8%) by PCR technique, where the GltA gene was amplified with an expected molecular size of 200 bp, as shown in Figure 1. From the blood, smears evaluated, E. canis was identified in only 41 of the samples evaluated (10.7%). Morulae were observed in the cytoplasm of lymphocytes and monocytes as round structures, with a size between 4 to 6 µm in diameter that stained strongly basophilic in color; as shown in Figure 2. On the other hand, none of dogs evaluated by PCR or smear evaluation was positive for A. phagocytophilum (Figure 3).
M) Molecular weight primer, (C+) positive control with 200 bp molecular weight, (115, 117,118, 119, 120, 121, 126) positive samples, (C-) negative control with double distilled water. 2% agarose gel, stained with Diamond.
Figure 1 PCR amplification of E. canis in blood samples taken from canines
Figure 2 Lymphocyte in peripheral blood of a canine infected with an E. canis morula (arrow). Giemsa stain 10%
Characteristics of the dog population
A number of 192 females (50%) and 192 males (50%), ranging in age from 3 months to 20 years, were evaluated. The observed results show that E. canis does not distinguish between genders, since within the infected group the percentages of females (29.7) and males (24.0) were not statistically significant (p>0.05). When evaluating the relationship between dog age (puppies, adults or seniors) and the percentage of E. canis positives, it was determined that there is a significant relationship between both variables, where the adult condition (1 to 7 years) is related to the presence of the disease (p<0.05) (Table 2).
Table 2 Frequencies and percentages of E. canis positives and negatives grouped by animal characteristic and season of year
| Presence of E. canis | Value of p | ||||
|---|---|---|---|---|---|
| Variable | Positive Frequency | % | Negative Frequency | % | |
| Sex | 0.205 | ||||
| Male | 42 | 24.0 | 146 | 76.0 | |
| Female | 57 | 29.7 | 135 | 70.3 | |
| Age | 0.016 | ||||
| Puppy (0-12 months) | 16 | 22.2 | 56 | 77.8 | |
| Adult (1 a 7 years) | 69 | 32.5 | 143 | 67.5 | |
| Senior (>7 years) | 18 | 18.0 | 82 | 82.0 | |
| Breed | 0.981 | ||||
| Crossbreed | 19 | 26.0 | 54 | 74.0 | |
| Pedigree | 84 | 27.0 | 227 | 73.0 | |
| Season of year | 0.816 | ||||
| Spring-Summer | 71 | 26.3 | 199 | 73.7 | |
| Fall-Winter | 32 | 28.1 | 82 | 71.9 |
Pedigreed dogs represented 81% (311/384) of the study population and crossbreed constituted 19% (73/384); however, the chi-square test of independence found no significant statistical difference between E. canis positive result, in relation to the defined racial groups and crossbreed (p>0.05) (table 2). Similarly, no significant differences were found between the presence of Ehrlichiosis and the year season (Table 2).
Hematological variables
In relation to laboratory findings for E. canis positive dogs, there were no significant differences in those showing anaemia, thrombocytopenia or hyperproteinemia compared to negative animals, many of which presented percentages similar to the infected group (p>0.05). On the other hand, significant differences were found for some white blood cell parameters, such as total leukocyte and neutrophil counts (p<0.05). However, for these analytes, the greater number of dogs infected with Ehrlichia were those that resulted with values within the reference ranges, in comparison with the animals that resulted negative where a large number of dogs with leukocytosis or neutrophilia are shown. For the rest of the hematological parameters evaluated, the statistical test did not find significant differences (p>0.05), as shown in Table 3.
Table 3 Frequencies and percentages of E. canis positives and negatives grouped in red series and platelets
| Presence of E. canis | Value of p | ||||
|---|---|---|---|---|---|
| Variable | Positive Frequency | % | Negative Frequency | % | |
| Hematocrit | 0.280 | ||||
| Anaemia (< 0.37 L/L) | 49 | 24.3 | 153 | 75.7 | |
| Without anaemia (≥0.37 L/L) | 54 | 29.7 | 128 | 70.3 | |
| Plasma Proteins | 0.739 | ||||
| Without hyperproteinemia (<75 g/L) | 45 | 25.7 | 130 | 74.3 | |
| With hyperproteinemia (>75 g/L) | 58 | 27.8 | 209 | 72.2 | |
| Platelets | 0.946 | ||||
| Thrombocytopenia (<180X109/L) | 6 | 28.6 | 15 | 71.4 | |
| Without thrombocytopenia (≥180X109/L) | 97 | 26.7 | 266 | 73.3 | |
| Leukocytes | 0.005 | ||||
| Leukopenia (<6x109/L) | 3 | 15.8 | 16 | 84.2 | |
| Normal (6-17x 109/L) | 71 | 33.3 | 142 | 66.7 | |
| Leukocytosis (>17x109/L) | 29 | 19.1 | 123 | 80.9 | |
| Monocytes | 0.060 | ||||
| Without Monocytosis (≤1.4x109/L) | 31 | 21.1 | 116 | 78.9 | |
| Monocytosis (>1.4x109/L) | 72 | 30.4 | 165 | 69.6 | |
| Lymphocytes | 0.235 | ||||
| Lymphocytosis (>4.8x109/L) | 12 | 18.5 | 53 | 81.5 | |
| Normal (1.0-4.8x109/L) | 72 | 28.1 | 184 | 71.9 | |
| Lymphopenia (<1.0x109/L) | 19 | 30.2 | 44 | 69.8 | |
| Segmented Neutrophils | 0.004 | ||||
| Neutropenia (<3.0 x109/L) | 30 | 18.4 | 133 | 81.6 | |
| Normal (3.0-11.5x109/L) | 70 | 33.8 | 137 | 66.2 | |
| Neutrophilia (>11.5x109/L) | 3 | 21.4 | 11 | 78.6 | |
| Eosinophils | 0.575 | ||||
| Without eosinophilia (<0.9x109/L) | 90 | 26.2 | 253 | 73.8 | |
| With eosinophilia (>0.9x109/L) | 13 | 31.7 | 28 | 68.3 |
DISCUSSION
Nowadays, canine ehrlichiosis and anaplasmosis have gained greater importance worldwide, which is mainly attributed to the fact that their vector (Rhipicephalus sanguineus) is considered the tick species with the widest geographical distribution (Aguiar et al., 2007;Parola et al., 2013; Cabezas-Cruz et al., 2019). In this research work, it was found that of the total number of dogs evaluated (384), 103 were positive for E. canis (26.8 %) by PCR technique and only 41 (10.7 %) through blood smear evaluation. This discrepancy of the two methods used is similar to that reported by Happi et al., (2018), who out of a total of116 dog samples only 10.3% were positive by microscopy, compared to the PCR technique where 42 positive results were obtained (36.2%). These results were to be expected, since although the diagnosis by microscopic visualization of the typical intracellular inclusions or morulae within the cytoplasm of monocytes or lymphocytes in peripheral blood smears (Figure 3) has been of great importance. This technique has certain disadvantages, such as lack of sensitivity during the early phase of infection, when there is low bacteremia, or when the bacterium multiplies in intracytoplasmic microcolonies in lymphoid organs. It will acquire mechanisms that ensure evasion of the immune response within the host cell (Bai et al., 2017;Manasa et al. 2017;McClure et al., 2017;Tominello et al., 2019;Franco-Zetina et al., 2019). In addition, false negatives have been reported in chronic or transient cases, because morulae usually disappear five to eight days after infection, as revealed in experimental studies in dogs and cattle (Gal et al., 2008;Stuen et al., 2013).
In Mexico, these diseases are frequently underdiagnosed, with few studies that determine their prevalence. In 2009 in Yucatan, a seroprevalence of canine ehrlichiosis of 45% was recorded (Jiménez-Coello et al., 2009) and in another investigation involving 28 states of the Mexican Republic. The presence of antibodies against Anaplasma spp, Borrelia burgdorferi and E. canis; registering a high prevalence for E. canis (55%) and moderate for Anaplasma spp (16.4%), for some northeastern states such as Coahuila and Nuevo
León (Movilla et al., 2016). Geographically, the animals that participated in this study belong to the northeastern zone of Mexico; however, if we compare the prevalence obtained in the central zone of Tamaulipas for E. canis (26.8%) with these two states, it would be much lower. However, it is important to mention that serological tests were used in this study, which may have the disadvantage of cross-reacting with other closely related microorganisms, overestimating the prevalence results and suggesting the need to carry out studies with molecular techniques that allow more accurate evidence of the type of pathogen involved (Cetinkaya et al., 2016).
In 2019, a molecular detection study of E. canis was conducted in rural areas of Yucatan, finding a 29.26% prevalence (Ojeda-Chi et al., 2019), which is close to that reported in this work (26.8%); but much higher compared to the prevalence found in dogs evaluated in the Comarca Lagunera (4%) (Almazán et al., 2016).
As for A. phagocytophilum infections, they have been increasingly diagnosed in companion and farm animals’ worldwide (McMahan et al., 2016). In Mexico, A. phagocytophilum, has been detected in opossums and dogs in Campeche state, with a prevalence of 3 and 27%, respectively (Rojero et al., 2017); however, in this work none of the dogs tested were positive by PCR or blood smear. This is not surprising, since Ixodes spp. and Dermacentor spp. ticks, infrequent in the study area, have been recognized as the most important vectors in the transmission cycle of this bacterium, which could have contributed to its null presence (Tinoco-García et al., 2009;Guzmán-Cornejo et al., 2016;Rodríguez-Vivas et al. 2019).
The results observed in this research show that E. canis has no predilection between gender, since within the infected group the percentages of females (29.7) and males (24.0) were not statistically significant (p>0.005). This same variable has been studied by several authors (Nuñez, 2003;Rodríguez-Vivas et al., 2005), finding similar results. However, this disagrees with what has been reported by other researchers, where they argue that females, especially during estrus, pregnancy or parturition, favor the risk of contracting E. canis infections (Salazar et al., 2014;Abdelfattah et al., 2021).
In relation to hematological findings associated with the presence of canine ehrlichiosis and anaplasmosis, it has been reported that these alterations will depend on the disease stage (Afusat et al., 2020). During the acute stage, the presence of anaemia is common, which is usually mild to moderate (usually normocytic, normochromic, non-regenerative) (Eberts et al., 2011).
In this work, the presence of anaemia was not significantly related to any of the diseases. Thrombocytopenia has been a hematological finding that has traditionally been associated with canine ehrlichiosis (Piratae et al., 2019). However, in this study the presence of thrombocytopenia (<200,000) had no association with E. canis positive animals. Several studies have reported an association between platelet count and the presence of E. canis, particularly in animals with platelet cell counts below 100 X109/L (Bulla et al., 2004; Tngsahuan et al., 2020). Although in the study many animals were reported with the presence of anaemia and hyperproteinemia, there is no significant statistical association when compared with animals that tested negative. This may be due to the possible presence of other hemoparasites such as Ehrlichia ewingii or Anaplasma platys that can produce degrees of anaemia and hyperproteinemia similar to those reported in dogs infected with E. canis (Piratae et al., 2019).
On the other hand, it is possible that many of the E. canis-positive individuals with unaltered hematological results had been in the subclinical phase of the disease. The latter would be of great importance since if the disease is not detected during this phase it could progress to a chronic stage, producing severe irreversible damage such as thrombocytopenia, leukopenia and severe non-regenerative anaemia resulting from bone marrow suppression (Little et al., 2014).
Regarding the evaluation of the white series, it is observed that despite the existence of significant differences between negative and positive cases to E. canis for total leukocyte, neutrophil and monocyte counts; the results were not as expected, since the negative dogs resulted with more alterations in these cells (either increased or decreased), compared to the positive ones. These findings are in agreement with the results obtained by Asgarali and colaboradores (2012), who reported that dogs with Ehrlichiosis manifested neutrophil and monocyte levels within reference ranges; in contrast to negative animals, which had a significant increase in these cells. A possible explanation for why many of the positive dogs showed no alterations in the white series is that these animals may have been in the subclinical phase of the disease, where most of them are asymptomatic and do not present significant hematological alterations (de Castro et al., 2004).
CONCLUSIONS
The present study showed that the hematological alterations evaluated in dogs with suspicious signs of Ehrlichia canis were not specific, since a large number of these animals were not infected. On the other hand, many of the dogs that did test positive remained without apparent changes in their blood counts, which is of great relevance, since these individuals, if not diagnosed in time, could be reservoirs for other hosts including humans. In addition, the veterinary clinician should consider that these diseases could present a subclinical picture without signs or with the presence of co-infections that produce similar signs, which would hinder their diagnosis and therefore the adequate treatment. Further research is suggested that includes the detection of other species of hemoparasites in the region, due to their importance as potentially zoonotic agents.
ACKNOWLEDGMENTS
To PRODEP Project No. 511-6/2019.-13905 entitled "Molecular evidence of tick-borne pathogens and their association with hematological changes in naturally infected canines in Cd. Victoria, Tam., Mexico" awarded by the promotion to Generation and Innovative Application of Knowledge (GAC) as part of the Support for the reincorporation of Former Scholarship Holders.
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Received: March 09, 2021; Accepted: June 14, 2021
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