Research Article | Open Access
Luma FM. Al-Ethafa1 , Ahlam AS. Al-Galebi2 and Mithal KA. Al-Hassani2
1Environmental Health Department, College of Environmental Science, Al-Qasim Green University, Iraq.
 2Department of Biological Science, College of Education, Al-Qadisiyah University, Iraq.
J Pure Appl Microbiol, 2019, 13 (3): 1745-1751 | Article Number: 5625
https://doi.org/10.22207/JPAM.13.3.49 | © The Author(s). 2019
Received: 17/05/2019 | Accepted: 22/07/2019 | Published: 15/09/2019
Abstract

The aim of the present study was to detect the prevalence of Anaplasma marginale in buffaloes in two Iraqi governorates, Al-Qadisiyah and Babylon, by the microscopy as well as the competitive-ELISA that used firstly among the Iraqi buffaloes. A total of 184 buffaloes from both sexes of different age groups of animals were submitted for collection of blood samples to prepare the smears and sera. Overall results were revealed on 10.33% and 36.41% positive animals by microscopy and competitive-ELISA, respectively. In addition, positive rates by both tests were 7.61%; by microscopy only, 2.72%; and by competitive-ELISA only, 28.8%. In Al-Qadisiyah and Babylon governorates, respectively, 8.7% and 11.96% of microscopy samples, and 44.57% and 28.26% of competitive-ELISA were positives with significant differences (P>0.05). Regarding to age factor, the highest prevalence was detected by microscopy in young age group (1-3 years) was 14.15%, whereas by competitive-ELISA, it was 55% in adult buffaloes group (>3 years). Significant increases (P>0.05) in rates of infection were showed in females compared to males, respectively, by microscopy (11.18% and 4.35%) and competitive-ELISA (39.13% and 17.39%).

Keywords

Anaplasma marginale, Buffaloes, Microscopy, Competitive-ELISA, Iraq.

Introduction

Anaplasma marginale is rickettsial intra-erythrocytic organism that causes bovine anaplasmosis in tropical, sub-tropical and temperate countries of the world, including Iraq; and being endemic in most animals of these regions1,2. Anaplasma that classified in Alphaproteobacteria class, Rickettsiales order of Anaplasmataceae family, is transmitted to cattle biologically by ticks and mechanically by flies and fomites3,4. Clinical disease is most notable in cattle, but other ruminants including water buffaloes can become persistently infected with A. marginale5. During acute anaplasmosis, A. marginale invades and multiplies within mature erythrocytes, leading to variable degrees of hemolytic anemia, fever, anorexia, weight loss, decreasing in milk production, reproductive problems and death in some cases6, 7. Recovering from acute phase results in persistent infected animal that serve as long-term reservoirs for transmission of infection within a herd8. The disease is a major constraint to bovine production because it affects dairy and beef domestic ruminants at any age resulting in high economic losses that estimated to be over 300 million dollars per year in United States 9, 10.

Microscopy is easy to perform, inexpensive, and considered as a “gold standard” test for confirming the acute phase of disease; however, its labor intensive and tedious for large numbers of samples, less sensitive, and impractical for routine testing of persistently infected ruminants as the bacterium is seldom detected in this phase11, 12. Hence, many serological techniques have been developed to detect specific IgM and IgG antibodies such as complement fixation test (CFT), card-agglutination (CAT), immunofluorescent antibody (IFAT) and enzyme-linked immunosorbent assay (ELISA)13, 14. Competitive-ELISA based on monoclonal antibody to recognize the major surface protein 5 (MSP5) of A. marginale, is used currently for diagnosis of bovine anaplasmosis15. It is highly accurate in diagnosis of acute and chronic infections with sensitivity and specificity that can reach to 95.6% and 98.6% of, respectively16, 17.

The purpose of this study was to evaluate the prevalence A. marginale infections in buffaloes through microscopic diagnosis of intra-erythrocytic A. marginale-inclusion bodies in slides of blood smears, and serological detection of specific anti-A. marginale antibodies in sera, for first time in Iraq, by a competitive-ELISA. In addition, the association of positive samples obtained by both assays to some epidemiological factors (residence, age, sex) of study’s buffaloes was evaluated.

Materials and Methods

Study’s samples
This study was performed in some rural regions related for two Iraqi governorates, Al-Qadisiyah and Babylon, during the period of March to August 2017. A total of 184 buffaloes from both sexes and different age groups were selected for the present study. From each animal, 10 ml of jugular venous blood was drawn and divided into two tubes (AFMA, Jordan); 3ml within an EDTA-anticoagulant tube to prepare of blood smear, and 7 ml within a free-anticoagulant tube that centrifuged at 3000 rpm for 15 minutes for sera. All sera were saved into numbered 1ml eppendorf tubes (China) and frozen at -20°C until be used18.

Blood smears (Preparation and examination)
Acutely infected buffaloes with A. marginale were diagnosed by using a rapid staining of Diff-Quick set (Modified Giemsa). According to manufacturer instructions (Vetlab Supplies, United Kingdom), the slides of thin blood smears were prepared, fixed with fixative solution, stained with solution I then solution II, rinsed with distilled water and, finally, dried by air. By light microscope (Trinoculr-MEIJI, Japan), the stained slides were examined under oil immersion to detect the positive samples that having intra-erythrocytic corpuscles of A. marginale as small dark spots, of peripheral location, and ranging from 0.1-0.8 mm19, 20.

Serological Survey
Competitive-ELISA was established for detection of specific IgG antibodies in persistently infected buffaloes with A. marginale. According to manufacturer instructions (VMRD, USA), the sera tested, and the results read using a microplate absorbance spectrophotometer reader (BioTekג, USA) at an optical density (OD) of 650nm. The test validation has been made as the mean of negative control must have an OD>0.40 and ≤2.10, whereas, mean of positive control must have an inhibition of ≥30%. Regarding to interpretation of samples values, samples having ≥30% inhibition rate were considered positive.

Statistical analysis
All obtained data were tabled and classified using of Microsoft Office Excel program (2013), and analyzed by a computerized IBM/SPSS program (V.23) through application of descriptive statistics and Chi-square test (x2). The significant differences between positive results of microscopic and serologic assays, and within residence, age, and sex factors of study’s animals, were compared and detected at a level of P≤0.0521.

RESULTS

Microscopic examination of blood smears obtained from 184 study’s buffaloes revealed that 19 (10.33%) buffaloes were positives with specific intra-erythrocytic inclusion bodies of A. marginale, (Fig. 1). In addition, sera samples of 184 buffaloes were tested by a serologic competitive-ELISA that detected 67 (36.41%) seropositive buffaloes with anti-A. marginale IgG antibodies, (Table 1).

Fig. 1. Intra-erythrocytic A. marginale inclusion bodies

Table (1):
Prevalence of A. marginale in an overall 184 buffaloes

Test No. Positives Negatives
Light Microscope 184 19 (10.33%) B 165 (89.67%)
Competitive-ELISA 67 (36.41%) A 117 (63.59%)

Variation in large letters, vertically, refers to significant differences at level of P≤0.05

The results of (Table 2) showed that 14/184 (7.61%) of buffaloes were positives by both microscopy and competitive-ELISA, and 112/184 (60.87%) were negatives by both tests. On other hand, 5/184 (2.72%) of buffaloes were positives with microscopy, only; whereas, 53/184 (28.8%) were positives by competitive-ELISA, only.

Table (2):
Cross-classification results of microscopy and competitive-ELISA

                                  Competitive-ELISA Total
Microscopy Positives Negatives
Positives 14 (7.61%)Ba 5 (2.72%)Bb 19
Negatives 53 (28.8%)Ab 112 (60.87%)Aa 165
Total 67 117 184

Variation in large vertical and small horizontal letters refers to significant differences

Animals of this study were comprised 92 buffaloes from some areas of each governorate. Whereas, 11 (11.96%) and 41 (44.57%) of buffaloes were positives, respectively, by microscopy and competitive-ELISA in Al-Qadisiyah; 8 (8.7%) and 26 (28.26%) were positives by both tests, respectively, in Babylon (Table 3).

Table (3):
Association of positive A. marginale infections to residence factor

Residence No. Microscopy Competitive-ELISA
Al-Qadisiyah 92 11 (11.96%) Ab 41 (44.57%) Aa
Babylon 92 8 (8.7%) Ab 26 (28.26%) Ba
Total 184 19 67

Variation in large vertical and small horizontal letters refers to significant differences

Among three age groups, positive buffaloes of microscopy and competitive-ELISA were distributed, respectively, as follow: in <1 year age group, 1/38 (2.63%) and 6/38 (15.79%); 1-3 years age group, 15/106 (14.15%) and 39/106 (36.79%); and in >3 years age group, 3/40 (7.5%) and 22/40 (55%), (Table 4).

Table (4):
Association of positive A. marginale infections to age factor/span>

Age No. Microscopy CompetitiveELISA
<1year 38 1 (2.63%) Cb 6 (15.79%) Ca
1-3 years 106 15 (14.15%) Ab 39 (36.79%) Ba
>3 years 40 3 (7.5%) Bb 22 (55%) Aa
Total 184 19 67

Variation in large vertical and small horizontal letters refers to significant differences

Among 161 female buffaloes, 18 (11.18%) and 63 (39.13%) were positives by microscopy and competitive-ELISA; while in 23 males, 1 (4.35%) and 4 (17.39%) were positives by both diagnostic methods, respectively, (Table 5).

Table (5):
Association of positive A. marginale infections to sex factor

<th “>CompetitiveELISA

Sex No. Microscopy
Female 161 18 (11.18%) Ab 63 (39.13%) Aa
Male 23 1 (4.35%) Bb 4 (17.39%) Ba
Total 184 19 67

Variation in large vertical and small horizontal letters refers to significant differences

DISCUSSION

According to FAO report in 1997, buffaloes are recognized as the “Black gold of Asia”, however, few neglected studies have examined the occurrence of A. marginale among buffaloes if compared to other field animals22. In this study, the total rate of positive buffaloes with A. marginale was 10.33% by slides of blood smears microscopy and 36.41% by serological competitive-ELISA (Table1). In previous studies, the occurrence rate of A. marginale among buffaloes by blood smears microscopy was reported 5.71% in Iraq23, 10.3% in Philippines (24), 4.29-22% in Pakistan22, 25, 33.5% in South Africa26, 59.3% in Egypt27; whereas, the seroprevalence of anti-A. marginale antibodies among buffaloes was 63% in Brazil28, and 78.1% in Egypt27. Also, the study reported that 2.72% of buffaloes were positives, only, by light microscopy, which might be explained by the persistence of recent infection and IgG-antibodies were not developed, completely, to be detected by competitive-ELISA29; whereas, 7.61% of buffaloes were positives by both tests, which can be explained that these animals with acute infection and have high level of IgG-antibodies from previous exposure30, at late stage of acute infection where the number of parasitemia decreased clearly and the immunity was increased, drastically31, or presence of high immunity with severe infection4. Major surface protein (MSP5) is a highly conserved surface protein among different strains of A. marginale, which has been proven as effective diagnostic antigen and used in a competitive-ELISA32. MSP5-competitive-ELISA demonstrated a high sensitivity and specificity for determining the true-positive and true-negative animals (bovine, ovine, caprine, camelidae) in endemic areas2, 33. In addition, the test is excellent for detection of specific IgG antibodies in sera of naturally or experimentally infected hosts and in vaccinated animals, so that, it can be applied for eradication programs, regulation of interstate and international movement of reproductive field hosts15,34. Many studies reported that the test has an ability to detect of individually infected animals accurately. Hence, it can be utilized for epidemiological investigations where the infections expanding through the movement of infected animals into disease-free regions35, 36.

Although, the worldwide seroprevalence of bovine anaplasmosis in buffaloes was reported to be less than that detected in cattle, the seropositive results of this study were higher than those reported previously in Iraqi cattle by2, 37. This could be attributed to that study’s buffaloes were exposed for unsuitable environmental conditions such as stress factors and ticks38. Other reasons are the bad management systems that include problems in feeding, drinking, housing and disease control or medication, which leading to decrease or waning of immunity. In general, buffaloes can play a role for harboring A. marginale and act as a potent carrier for other animals39, 40.

In microscopy, although the positive prevalence of buffalo’s A. marginale in Al-Qadisiyah (11.96%) was higher than reported in Babylon (8.7%) governorates; no significant differences (P£0.05) have been detected relatively between them, (Table 3). Whereas, the seroprevalence of infection by competitive-ELISA in Al-Qadisiyah (44.57%) much more than showed in Babylon (28.26%), and this could belong to variations in either owner’s subculture, topography or to some risk factors such as stocking density, type of dipping, introduction of cattle to the farm, farm type, herd size, tick density, and dipping intervals41, 42.

Positive results among different buffalo’s age groups (Table 4) detected that the highest prevalence by microscopy was showed in young buffaloes (1-3 years age group), whereas by competitive-ELISA, it’s seen in adults (>3 years age group). In young animals, these results might be explained by the age resistance and lack of maternal immunity gained by colostrums, which may last up 6 months to 1 year, hence more exposure for infections; whereas in adults, the seroprevalence of IgG-anti A. marginale antibodies was interpreted by the facts that the disease is of adults and the high titer levels of antibodies might be reflection for previous frequent multiple exposure to Anaplasma or recent infection 26, 27.

In relation to sex factor (Table 5), significant increases in A. marginale infections were detected in female buffaloes by both the microscopy and competitive-ELISA, which might belong to the low samples of study males in comparison to females, exposing of females to high stress conditions (gestation, parturition, milking), and/or that males received an attention more than females concerned to housing, feeding and medication43, 44, 45.

CONCLUSION

The present study resumed that the prevalence of A. marginale in buffaloes have been increased, clearly, when compared to previous Iraqi studies; as well as, the seropositive results by competitive-ELISA were much more than reported by microscopy. In addition, differences in positivity among residence, age and sex factors could provide a benefit data for a futurism studies that recommended to be depended on competitive-ELISA or molecular techniques as a high sensitive and specific diagnostic methods.

Declarations

Acknowledgements
None.

Conflicts of Interests
The author declare that there are no conflicts of interest.

Authors’ Contribution
Author listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

Funding
None

Data Availability
All datasets generated or analyzed during this study are included in the manuscript.

Ethics Statement
This article does not contain any studies with human participants or animals performed by any of the authors.

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