Serological Investigation of Bovine Chlamydia abortus in Wasit Province, Iraq

1. Introduction

Chlamydia abortus is a non-motile, coccoid, pleomorphic, gram-negative, obligatory intracellular bacterium that belongs to the Chlamydiaceae family of the Chlamydiales Order classified under the phylum of Chlamydiota ( 1 ). Chlamydia spp. has a unique life cycle, including the non-infectious reticulate form found only inside the cell and the smaller and relatively inert infectious elementary form taken by a host cell and remains inside a membrane-bound inclusion body in the cytoplasm of a cell ( 2 ). Direct and indirect infection transmissions have been reported as a result of the excretion of organisms in large amounts by the expelled placenta, uterine discharges, and feces of infected and aborted animals ( 3 ). In many countries around the world, C. abortus is a cause of abortion and fetal losses, mainly in mammals, in particular ruminants (cattle, sheep, and goats) and non-mammalian hosts, causing tremendous economic losses ( 4 - 6 ).

In Iraq, the incidence of reproductive diseases in cattle appears to be increasing over the years. Chlamydia diseases are frequently asymptomatic, and clinical signs are seen individually in cattle, often noticed as a non-specific loss in reproduction, sub-clinical mastitis, pneumonia, and weight loss ( 7 ). Also, most adult animals that have been infected before pregnancy show no clinical signs of infection, with the organism arriving in a dormant phase ( 8 ). In pregnant cows, C. abortus infection occurs during the 6^th to 8^th months of gestation, particularly among heifers in their first pregnancy, sometimes resulting in weak and premature calves ( 9 , 10 ). In aborted cases, a presumptive diagnosis can be made based on gross pathological lesions in cotyledons and intercotyledonary membranes and confirmed by either detection of elementary bodies in stained smears ( 11 ) or isolation of organisms by culture ( 12 ). However, both methods cannot be applied in epidemiological studies due to difficulties in isolation, requiring high expertise and special laboratories ( 13 ). Other methods include antigen detection using immunochemical and molecular assays and antibody detection using serological techniques, characterized by their high sensitivity and specificity ( 14 ). The Enzyme-linked immunosorbent assay (ELISA) is one of the most specific available tests and is usually applied in epidemiological surveys ( 15 ).

The serological prevalence of C. abortus varies considerably between regions of a country as well as between different countries ( 7 , 16 ). Due to the zoonotic potential, C. abortus has attracted increasing interest. However, available data about the prevalence of the organism in cattle in Iraq remains low and needs to be supported using advanced diagnostic assays. Therefore, for the first time, this study was designed to investigate the seroprevalence of C. abortus in cattle in Wasit province (Iraq) and to identify the severity of infection among seropositive animals. Additionally, our study aimed to estimate the association of positive findings with risk factors, age, sex, and region.

2. Materials and Methods

2.1. Study Animals

Two hundred seventy-six cattle of different ages and sexes were selected randomly from different areas (four regions, Al-Kut, Al-Hai, Al-Numaniyah, Sheikh Saad) in Wasit province (Iraq) from November (2021) to January (2022). Each study animal was subjected to draining jugular venous blood under aseptic conditions into vacuum-free-anticoagulant glass tubes centrifuged at 5000 rpm (5 minutes). The sera were transferred into labelled Eppendorf tubes and frozen in a refrigerator at 4 ºC until examined serologically by ELISA.

2.2. Serological Examination by ELISA

Following the manufacturers' instructions for the Bovine Chlamydia abortus ELISA Kit (SunLong Biotech, China), the serum samples, solutions, and micro ELISA strip plate were prepared. For each ELISA kit, the procedure involved adding 50l of positive and negative control solutions, in duplicate, to the first wells (1-4) with adding 40l of Sample Dilution Buffer and 10l of sera to all other wells (5-95) except the blank well (96). The microplate was sealed, incubated (37ºC / 30 min), and washed with the Diluted Washing Buffer 5 times. A total o∅ 50 μl of HRP-Conjugate was added to all wells except the blank, and the microplate was sealed, incubated (37ºC / 30 min), and washed with the Diluted Washing Buffer 5 times. A total o∅ 50 μl of each Chromogen A and B was added to each well (except the blank), and the microplate was incubated (37ºC / 15 min). A total o∅ 50 μl of Stop Solution was added to all wells, and the microplate was placed in the Microtiter Plate Reader (BioTek, USA) to measure absorbance at an optical density (OD) of 450nm. For determining results, effectiveness identification, critical value (CUT OFF) calculation, and positive/negative judgments were determined at <253 for negative OD and ≥253 for positive OD. Based on their values, the positive ODs were classified into 3 categories; mild (253-450), moderate (451-600) and severe (>600) infections.

2.3. Statistical Analysis

All obtained data were documented using the Microsoft Office Excel version 2016 (Microsoft, USA) software and analyzed statistically using GraphPad Prism version 9.0.2 (GraphPad Inc, USA). One-Way ANOVA and Odds-Ratio were applied to detect significant differences between groups in the severity of infections and to estimate the association between seropositivity and risk factors (including age, sex and region), respectively. Each value was represented as either a percentage (%) or MeanStandard Error (MSE). Variation was considered significant at P<0.05.

3. Results

The findings of ELISA results revealed 17.03% (47/276) positive samples that involved 63.83% (30/47) mild, 25.53% (12/47) moderate and 10.64% (5/47) severe infections (Figures 1 and 2). In addition, values of mild, moderate and severe infections were 0.32710.0085, 0.5270.0139 and 0.70840.0256, respectively (Figure 3).

Figure 1. Total results of testing of Chlamydia abortus infection in 276 cattle by ELISA(P≤0.0017) **

Figure 2. Distribution of percentage of positive ODsaccording to the severity of Chlamydia abortus infection. (P<0.05) *

Figure 3. Distribution of values of positive ODs according to severity Chlamydia abortus infection(P≤0.0009) ***, (P≤0.0001) ****

Concerning risk factors, age, sex and region, the prevalence of positive findings was significantly variable (Table 1). For age, significant increases (P<0.043) in the prevalence of C. abortus were detected in cattle aged < 3 years (31.71%) in comparison with 3-6 (15.46%) and >6 (13.77%) groups that both showed insignificant differences (P>0.05) in their values. Also, cattle of < 3 years appeared at a significantly higher risk of infection (2.19) than 3-6 (0.86) and >6 (0.68). Regarding sex, there were significant increases (P<0.034) in prevalence and risk exposure of females (19.56% and 3.33, respectively) to C. abortus when compared to males (5.88% and 0.3, respectively). For the region, significantly higher prevalence and risk exposure to C. abortus were reported in Al-Hai (33.33% and 2.87, respectively), while lowered in Al-Kut (4.35% and 0.2, respectively) when compared to Al-Numaniyah (13.04% and 0.71, respectively) and Sheikh Saad (17.39% and 1.03, respectively).

Regarding the association between risk factors and the level of severity of infection, results reported a significant variation (P<0.05) in their values (Table 2). In this study, we found that cattle of <3 years showed a significant elevation (P<0.04) in moderate (46.15%) and severe (38.46%) C. abortus infection when compared to mild (7.69%) infection; however, cattle aged 3-6 and >6 years showed a significant increase of mild infection (73.33% and 94.74%, respectively) when compared to moderate (36.36% and 5.62%, respectively) infection. Additionally, no severe positive infections were seen in the 3-6 and >6 age groups. In females and males, mild infection (63.64% and 66.67%, respectively) demonstrated a significant (P<0.05) higher prevalence than moderate (27.27% and 0%, respectively) and severe (9.09% and 33.33%, respectively) infections. Among all study regions, mild seropositive C. abortus infection findings were significantly more significant than moderate and severe infections (P<0.05).

4. Discussion

The cattle industry had several challenges that affected its further development. Reproductive disorders represent one of these challenges worldwide and may be due to intrinsic and extrinsic factors imposed on the herd and individual animals, such as genotyping traits, feeding, contaminants and toxins in feeds or other environmental factors ( 17 ). Chlamydia abortus is one of the most common infectious bacteria with serious effects for infected pregnant animals and humans as well as for their fetuses. In this study, 17.03% of study cattle were positive serologically to C. abortus. In Iraq, only one recent study in Ninevah province detected that the seroprevalence of C. abortus in cattle was 0.82% ( 18 ).Worldwide, the seroprevalence of C. abortus in cattle was 1.69% in Belgium ( 19 ), 4.44% in Ireland ( 20 ), 4.65% in India ( 21 ), 17.83% in China ( 22 ), 26.4% in Poland ( 23 ), 26.92% in Turkey ( 24 ), 45% in Australia ( 25 ), 48.4% in Iran ( 26 ) and 51.3% in Taiwan ( 27 ). This variation in the prevalence of C. abortus between our study and others might be attributed to different factors such as the virulence of chlamydial strains and possibly innate immunity amongst animals, frequent exposure of study animals to infected or carrier animals, uncontrolled restriction for movement of diseased cattle from the contaminated region, nutritional deficiency, bad management, grazing strategies, breed of cattle, size of examined samples, type of serological test and its efficacy (sensitivity and specificity), and geographical location of the study. Significant prevalence of mild infection among seropositive cattle may be indicated that cattle are either less sensitive to being infected with C. abortus, increasing resistance to infection with advancing age as most study animals were larger than 3 years, or intracellular pathway of the organism which allows to limited exposure and then less developed antibody against infection.

Statistical analysis of risk factors (age, sex and region) revealed a significant difference in the distribution of seropositivity among these factors. For age, our findings demonstrated that cattle aged <3 Years were having a higher positivity rate of C. abortus infection than other age groups (3-6 and > 6 years) significantly, suggesting that C. abortus is either transferred vertically during pregnancy (inutero infection), their origin is due to subclinical mastitis of Chlamydia or due to reduced maternal immunity, especially after one year of age. ( 28 ). In this study, the significant prevalence of positivity in females may be caused by frequent infection by organisms due to its reproductive role and lowered immunity resulting from high stress due to gestation, milk production, and nutritional deficiency. Also, in this study, a low number of tested males and the fact that in Iraq, male cattle received more quantitatively and qualitatively nutritional and managemental attention since male calves were used for slaughter and for insemination purposes. Regarding study regions, cattle of the Al-Hai district showed a higher prevalence of C. abortus than in other regions. This might be due to the fact that animals in this region are mainly exposed to organisms, reduced therapeutic and controlled measures or direct contact with the carriers/reservoirs. The association of risk factors to the level of severity of infection confirmed that moderate and severe infections were more prevalent among cattle aged <3 Years; while mild infection appeared significantly in cattle aged 3-6 and >6 years, both females and males, and among all positive cattle throughout all study regions as reported by different studies as the calves are highly susceptible to infectious pathogenic agents, particularly newborn calves that not received adequate amounts of colostrums ( 18 , 29 ). This study showed that C. abortus is widespread in cattle, particularly adult lactating cows, which might represent a potential source of transmission of infection to humans. The classification of positive infections according to their ODs might provide a valuable framework for subsequent investigations of potential farm-level influences causing these larger-than-expected differences in herd antibody seroprevalence to C. abortus. While eradication of C. abortus in Iraq is unlikely, the most substantial and cost-effective method for reducing its impact could be achieved by targeting control measures such as vaccination, particularly in areas with high seropositivity or cattle with a history of high abortion rates. Cattle might be susceptible to C. abortus even if they do not, in general, exhibit disease, and the causes of chronic sub-clinical reproductive problems linked with infertility and mastitis should be studied precisely.

Authors' Contribution

Study concept and design: H. H. E. A.

Acquisition of data: H. A. N.

Analysis and interpretation of data: I. M. A.

Drafting of the manuscript: I. M. A.

Critical revision of the manuscript for important intellectual content: H. H. E. A.

Statistical analysis: H. A. N.

Administrative, technical, and material support: H. H. E. A.

Ethics

The current study was accepted by and carried out under the authorization of the Scientific Committee in the College of Alkut University (Wasit, Iraq) and by the College of Veterinary Medicine, University of Basrah (Basrah, Iraq).

Conflict of Interest

The authors declare that they have no conflict of interest.

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