Serological Evidence of Chlamydiosis, Q Fever and Brucellosis in District Dadu, Pakistan
Hubdar Ali Kolachi1, Shahid Hussain Abro1, Asghar Ali Kambhoh1,
Saeed Ahmed Soomro2*, Nazeer Hussian Kalhoro3 and Abdul Ahad Soomro4
1Department of Veterinary Microbiology, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, 70060 Tandojam, Pakistan.
2Department of Physiology and Biochemistry, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, 70060 Tandojam, Pakistan.
3Sindh Institute of Animal Health, Karachi, Pakistan.
4Central Veterinary Diagnostic Laboratory (CVDL), Tandojam Sindh, Pakistan.
ABSTRACT
Brucella melitensis, Chlamydophila (=Chlamydia) abortus and Coxiella burnetii are common abortifacient bacterial pathogens in sheep and goats. The establishment of active animal surveillance is essential to detect incidences as outbreaks of infections in small ruminants. The current study was planned to determine presence of these bacterial organisms causing abortions in goats and sheep in district Dadu, Sindh, Pakistan. A total of 224 samples (blood= 184, vaginal swab= 05, foetal membrane=02 and milk=33) were obtained from the selected animals. The health status, age, sex, parity and history of abortion for each goat and sheep was documented. The serological test I-ELISA and biochemical reactions were performed for detection of Brucella melitensis, Chlamydia abortus and Coxiella burnetii. The antibiogram of the isolated B. melitensis was performed using Kirby Baur Disk Diffusion method. On serological analysis of 184 blood samples, 118 (64.13%) samples were found positive for the abortifacient bacteria. Out of these 184 samples, 67 (36%), 47 (25%) and 4 (2.17%) samples were positive for C. abortus, C. burnetii and B. melitensis, respectively. Sixty-seven samples, caprine 34 (37.3%) and ovine 33 (35.3%), were found positive for C. abortus. Forty-seven samples, does 31 (33.33%) and ewes 16 (16.9%) were detected positive for C. burnetii. Only 3 (3.7%) samples from goats and 01 (1.5%) sheep samples were found positive to B. melitensis. There is no significant difference in the comparative prevalence of the three major abortive infection (C. burnetii, C. abortus and B. melitensis) among nulliparous, primiparous and multiparous goats and sheep. Out of 40 samples cultured (vaginal swab= 05, foetal membrane=02 and milk=33), 5 samples were found positive for B. melitensis. The antibiogram indicated B. melitensis isolates were highly susceptible to oxytetracycline, doxycycline and ciprofloxacin. While the organism found resistant to erythromycin, lincomycin, rifampicin, cephalothin, bacitracin, streptomycin and gentamycin. In conclusion, Brucella melitensis, Chlamydophila abortus and Coxiella burnetii infections were prevalent in different areas of district Dadu, Sindh, Pakistan. B. melitensis was highly sensitive to oxytetracycline, doxycycline and ciprofloxacin.
Article Information
Received 24 March 2021
Revised 19 July 2023
Accepted 11 August 2023
Available online 27 March 2024
(early access)
Published 30 May 2025
Authors’ Contribution
HAK carried out the experiment. SHA acted as a major supervisor of the project. AAK and SAS conceived the study design and analyse the data. NHK and AAS helped in writing of manuscript.
Key words
Q fever, Chlamydiosis, Brucellosis, Small ruminants, Sindh
DOI: https://dx.doi.org/10.17582/journal.pjz/20210324160335
* Corresponding author: [email protected]
0030-9923/2025/0004-1543 $ 9.00/00
Copyright 2025 by the authors. Licensee Zoological Society of Pakistan.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
INTRODUCTION
The first species of animals to be domesticated by human beings were sheep and goats. Sheep and goats constitute approximately half of the domesticated ruminants population in the world (Jesse et al., 2020). The global population of these animals have been increased from 1.35 billion to 1.94 billion (Solangi et al., 2023). About 90% of caprine farmed in the developing countries, are one of the valuable sources of protein for humans (Tyasi et al., 2020). In certain countries, small ruminants can be considered as “liquid asset” in h of need (Teklebrhan et al., 2014). According to economic survey of Pakistan, 2021-2022 contribution of livestock in agriculture sector is 61.9% and 14. 0% in overall GDP of Pakistan. Pakistan owns vast number of small ruminant population with an estimated number of 82.5 and 31.9 million heads of goats and sheep, respectively (Economic Survey of Pakistan, 2022).
Abortion in goats and sheep has worldwide importance because of crucial economic losses including zoonotic risk (Benkirane et al., 2015; Van Engelen et al., 2014). In the year 2016, 1275 miscarriage cases in sheep and 1143 in goats were reported in Jordan. Reproductive disorders have negative effect on farm animal outcomes, health and peoples economy (Chu et al., 2016; Van Engelen et al., 2014).
Various infectious and noninfectious agents are potential cause of abortion. Abortion due to pathogenic organisms usually takes place during the last three months of gestation (Holler, 2012). While noninfectious causes are phytotoxins, dietary deficiencies of micro minerals (copper, selenium, magnesium), vitamins, hormones (cortisol, estrogen), phenothiazine and CCL4 (Tramuta et al., 2011). There are number of infectious organisms such as Toxoplasma gondii, Brucella melitensis, Listeria spp., Chlamydia abortus, Coxiella burnetii, blue tongue virus, foot-and-mouth disease virus, small pox and Campylobacter fetus, which cause abortion in domesticated animals (Van den Brom et al., 2015; Ababneh et al., 2014; Abousenna et al., 2020; Jabary et al., 2020).
Chlamydophila (=Chlamydia) abortus, Brucella melitensis, and Coxiella burnetii are bacterial pathogens causing abortion (Ganter, 2015). In addition to these some other opportunistic pathogens also responsible for abortion (Vidal et al., 2016). The diseases caused by these organisms may lead to congenital defects, loss of foetus and abortion in ruminants (Van Engelen et al., 2014). Livestock owners in different districts of Sindh have reported higher abortion rates causing economic losses in small ruminants but the cause remained a mystery (Memon et al., 2022). Therefore, the establishment of active animal surveillance is essential to detect incidences as outbreaks of infections. Because of the impact of such infections on production and economic losses in livestock, the study was conducted to detect different bacterial organisms causing abortions in small ruminants and their prevalence in district Dadu, Sindh, Pakistan.
MATERIALS AND METHODS
The study design was based on abortion cases in the district Dadu, Sindh Pakistan, on the basis of the presence of susceptible clinical cases of abortion in small ruminants and previous evidence of abortions in goats and sheep. The study was conducted in ten villages of Dadu district that represented all four talukas of Dadu district viz., Dadu, Khaipur Nathan Shah, Mehar and Johi (Fig. 1). Dadu has rich alluvial loamy soil of the Indus valley and has high number of small ruminants, but overall Dadu district is economically backward area of Sindh province. Selection of villages was based on information collected through survey form. A total of 224 samples were taken from the selected animals using random-sampling approach. The health status, age, sex, parity and history of abortion for each goats and sheep was documented. In addition, the history of farming personals in relation to abortion, dystocia, infertility, still birth or any other reproductive disorder data was obtained.
In this investigation, a total of 224 (n=184 for serology and n= 40 for culture) samples were investigated for the presence of abortifacient bacteria. A total number of 112 goats were selected for sampling from Monder village, Dadu city, Khairpur Nathan Shah and Kakar areas. Whereas the 112 sheep samples were collected from in and around areas of Kakar and Mujaver villages of Khairpur Nathan Shah district Dadu. The samples were comprised of blood, milk, vaginal swab and placenta.
Collection of samples
Blood samples were collected from jugular vein of goats and sheep in 5ml sterile plain vacutainers aseptically. After collection samples were kept on ice and transported to the Department of Veterinary Microbiology, Sindh Agriculture University, Tandojam. The specimens were centrifuged at 10,000 rpm for 5 min to separate the sera. The sera were carefully transferred to sterile 3 ml Eppendorf tubes. The sera samples were stored at -20oC for further investigations.
Vaginal swab, foetal membrane and milk specimens were also collected from the ruminants with recent history of abortion or those aborted at the time of sampling in selected areas. Vaginal swabs samples were collected by culture swabs in sterile tubes containing transported medium sucrose phosphate glutamate (SPG) buffer and brought to the laboratory of Department of Veterianry Microbiology for further processing. Milk samples were aseptically collected from lactating goats and sheep by hand stripping using sterile screw capped 25ml tubes. The samples were transported to the Central Veterinary Diagnostic Laboratory Tandojam in ice bags and kept in refrigerator till processed for bacterial isolation using Brucella selective agar (Hi Media, India).
Serological assay
The serological test I-ELISA to detect Brucella melitensis, Chlamydia abortus and Coxiella burnetii was carried out at Sindh Poultry Vaccine Centre, Karachi, Sindh, using commercial kits of Enzyme linked immunosorbent assay (ELISA). All test procedures were carried out according to manufacturers’ instruction (IDEXX-Germany) for the use of B. melitensis (catalogue number P04130-10), C. burnetii (catalogue number QFT1135T) and C. abortus (catalogue number CLA1135T) kits.
Isolation of Brucella from milk and swab samples
Isolation and identification of Brucella was carried out as per OIE Manual Standard Procedure (2013). Forty clinically suspected samples of milk (n=33), placenta (n=2) and vaginal swab (n= 5) samples were inoculated on Brucella Selective Agar (Hi Media, India). The inoculated plates with specimen were incubated at 37oC for up to 4 days and colonies were checked after every 24 h. Brucella assumed colonies were picked up by sterile wire loop, mixed with a drop of distilled water on a clear glass slide to make smear. Then the smear was heat fixed and dried in an incubator. The slide was stained by Ziehl–Nielsen’s (differential stain) method. Brucella species identification was based on Gram negative, pin point smooth appearance and coccobacilli shape arranged mostly in single but some in pairs and in bunches according to Alton et al. (1988).
Antimicrobial susceptibility testing
The antibiogram of isolates was carried out using prescribed method of disk diffusion by Bauer et al. (1966). The bacterial standardized pure culture inoculum was swabbed onto surface of specific Mueller Hinton agar and Brucella selective agar. Filter paper disks impregnated with antimicrobial agents were placed on the media plates. After overnight incubation at 37 oC, the zones of inhibition were measured.
Statistical analysis
All collected data was entered Excel (Microsoft office, 365) spread sheets to calculate the means. Then it was transferred to statistix software (Version 8.1, 1985-2005 Analytical Software.) to calculate the risk factors using Chi-square test.
RESULTS
Sero-prevalence of the abortifacient bacteria in goats and sheep
On analysis of the 184 serum samples of ovine and caprine thorough I-ELISA, the total seroprevalence of common abortifacient bacteria obtained was 64.13%. 118 samples were found positive out of 184 samples (Table I). Individual pathogen wise, out of 184 serum samples 67 (36%), 47 (25%) and 4 (2.17%) were serologically positive for Chlamydia abortus, Coxiella burnetii and Brucella melitensis, respectively. The prevalence percentage of C. abortus found in does 34 (37.3%) was higher than ewes 33 (35.3%). Similarly, C. burnetii prevalence was higher in does 31 (33.33%) than ewes 16 (16.9%). Four samples (2.17%) were found positive for B. melitensis. Out of these four samples, 3 (3.7%) were detected positive from goats, while one sample (1.5%) from sheep was found positive (Table I).
Association of risk factors with abortifacient bacteria in small ruminants
The distribution of various abortifacient bacteria according to the parity (nulliparous vs primiparous vs multiparous) and abortion frequency (once vs ≥ twice) was analyzed and presented in Table II. There is no significant difference in the comparative prevalence of the three major abortive infection (C. burnetii, C. abortus and B. melitensis) among nulliparous, primiparous and multiparous goats and sheep. The Chi square calculations also showed no significant difference in these three abortive bacteria between once and ≥ twice aborted goats and sheep.
Brucella melitensis in milk and swab samples
In present study, Brucella melitensis was isolated from the milk and vaginal swabs. Out of total 40 samples 20% (01) from vaginal swab and 12.12% (04) from milk samples were found positive, while no isolates were obtained from placental samples of aborted animals (Table III).
Table I. Seroprevalence of abortifacient bacteria in goats and sheep in district Dadu, Sindh.
|
Animal species |
Samples tested |
Positive for C. burnetii |
Positive for C. abortus |
Positive for B. melitensis |
Total positive |
x2 (d.f) |
p-value |
|
Goats |
92 |
31 (33%) |
34 (37.3%) |
3 (3.7%) |
68 (73.91%) |
3.13(2) |
0.2092 |
|
Sheep |
92 |
16 (17%) |
33 (35.3%) |
1 (1.5%) |
50 (54.34%) |
||
|
Total |
184 |
47 (25%) |
67 (36%) |
4 (2.1%) |
Table II. The occurrence of abortifacient bacteria in relation to parity and abortion frequency in small ruminants.
|
Variable |
No. of animals |
C. burnetii |
C. abortus |
B. melitensis |
x2(d.f) |
p-value |
|
Parity |
||||||
|
Nulliparous |
26 |
4 (15.38) |
7 (26.92) |
0 (0) |
9.11 (4) |
0.06 |
|
Primiparous |
58 |
9 (15.51) |
10 (17.24) |
3 (5.17) |
||
|
Multiparous |
100 |
34 (34) |
50 (50) |
1 (1.00) |
||
|
Total |
184 |
47 |
67 |
04 |
||
|
Abortion frequency |
||||||
|
Once |
168 |
40 (23.8) |
58(34.52) |
03(1.78) |
0. 42(2) |
0.8087 |
|
≥ Twice |
16 |
7 (43.7) |
09(56.25) |
01(6.25) |
||
|
Total |
184 |
47 |
67 |
04 |
||
Table III. Isolation of Brucella melitensis from clinically suspected animals.
|
Sample type |
No. of samples cultured |
No. of isolates |
Percentage (%) |
|
Vaginal swab |
05 |
01 |
20% |
|
Placenta |
02 |
0 |
0% |
|
Milk |
33 |
04 |
12.12% |
|
Total |
40 |
5 |
12.5% |
Table IV. Antibiotic susceptibility of B. melitensis isolates from vaginal and milk samples of aborted animals.
|
Bacterial species |
Antibiotic discs used |
Inhibited zone diameter |
Degree of susceptibility |
|
B. melitensis |
Cefoxitin Oxytetracycline Lincomycin Erythromycin Doxycycline Rifampicin Cephalothin Streptomycin Bacitracin Ciprofloxacin Gentamycin |
10mm 24mm 00mm 00mm 21mm 00mm 00mm 00mm 00mm 21mm 00mm |
Partial sensitive Highly sensitive Resistant Resistant Highly sensitive Resistant Resistant Resistant Resistant Highly sensitive Resistant |
Antibiotic susceptibility profile of Brucella melitensis isolated from milk swab samples
B. melitensis species were identified based on microbiological and biochemical characteristics. The organism was subjected to a panel of eleven antimicrobials. The results indicating the efficacy of antibiotics against Brucella melitensis is presented in Table IV. The antimicrobial susceptibility test indicated that B. melitensis isolates was highly sensitive to oxytetracycline (24mm), doxycycline (21mm), ciprofloxacin (21mm) and partially sensitive to cefoxitin (10mm). While the organism found resistant to erythromycin, lincomycin, rifampicin, cephalothin, bacitracin, streptomycin and gentamycin.
DISCUSSION
This is the first study that was carried out in East part of Sindh (Dadu district) for investigation of Brucellosis, Chlamydiosis and Q fever prevalence. The objective to conduct this study was to collect essential information about the most commonly occurring etiologies of abortion in caprine and ovine in district Dadu of Sindh Province. Abortion in goats and sheep can be caused by several infectious agents. This study identified heavy burden of chlamydiosis in goats and sheep with high susceptibility in different areas of district Dadu Sindh. The present study indicated that among all three common abortifacient bacteria there was highest prevalence rate of Q fever after Chlamydiosis. This survey showed generally low prevalence of Brucella melitensis (Brucellosis) in the district Dadu, Sindh, Pakistan.
Q fever outbreaks in Netherlands related with sheep and goats have strengthen awareness of the disease as an important emerging zoonoses (Georgiev et al., 2013). Our study results exhibited that Q fever has high prevalence rate (25%) in the ovine and caprine population of study area. This finding is in accordance with studies of Zahid et al. (2016) who reported the highest prevalence of Q fever (30.8%) infection in goats and sheep of Punjab, Pakistan. While, Shabbir et al. (2016) reported 17.9% and 16.4% sero-prevalence of Q fever in goats and sheep, respectively. The literature reports only few studies on the prevalence of Q fever in country Pakistan (Kaplan and Bertagna, 1955; Ahmed, 1987; Zahid et al., 2016; Shabbir et al., 2016; Rashid et al., 2019; Memon et al., 2022). However, Kshash (2012) and Anastacio et al. (2013) have reported higher prevalence in sheep than goats. In the current study 224 animals (n=112 goats and n=112 sheep) were investigated. Seroprevalence of Q fever determined in two species was higher in goats (33.6%) than in sheep (17.3%). This corelates with the results of Van den Brom et al. (2015) and Zahid et al. (2016). An overall prevalence of Q fever was 25.5%. These findings agreed with earlier studied Q fever seroprevalence in goats and sheep of Iran, Turkey and the Netherlands (Esmaeili et al., 2014; Arserim et al., 2011; Schimmer et al., 2011). Rahman et al. (2018) reported seroprevalence of 6.38% in domestic ruminants of Bangladesh. Rashid et al. (2019) have reported seroprevalence of 6.1% in cattle and buffaloes in institutional farms of Punjab, Pakistan.
Brucellosis is not only a disease of zoonotic importance but also it has economic and productive importance. The disease highly affects reproductive and productive potential of animals (Shabbir et al., 2013). In this study total seroprevalence of brucellosis in caprine and ovine was noted to be 2.17%. Our findings regarding occurrence of Brucella infections are close to the last reports (Shafee et al., 2016; Hussain et al., 2014; Iqbal et al., 2013), who have reported 2.3%, 7% and 10% prevalence in Baluchistan, the Punjab and Kohat, respectively. This study is in contrast to the reports of Gul et al. (2015) who have seen 1.91% and 2% prevalence in sheep and goats in Pakistan. In Punjab, Pakistan Shahzad (2017) has reported prevalence of 0.23% and 0% in goats and sheep, respectively. Shafy et al. (2016) and Rahman et al. (2011) reported sero-prevalence of 9.53% and 2.6% in Bangladesh. An overall seroprevalence of 1.72% of caprine brucellosis has been documented in India (Saikia et al., 2019). In Nepal, B. melitensis prevalence in caprine was found to be 2.6% (Pandeya et al., 2016).
In the current study, Chlamydia abortus was isolated from specimens belonging to goats and sheep with the history of recent abortion. There are reports of Chlamydia infection from small ruminants in the world (Ababneh et al., 2014; Berri et al., 2009). However, there is lack of studies on Chlamydia infection in sheep and goats in Pakistan. The first chlamydial isolation from birds have been reported in 2011 (Esmaeili et al., 2017). The studies of Esmaeili et al. (2017) corelate with our studies, who reported that 25% samples were positive for chlamydiosis. In the Middle Atlas and Northen Morocco Benkirane et al. (2015) conducted serological survey of 13 flocks of sheep and found all (100%) positive, while out of 10 flocks of goats 8 (80%) were seropositive. As indicated by the monetary significance of chlamydial abortion and its zoonotic implications. In the present investigation, detection of Chlamydia affirmed the ovine enzootic abortion disease in the examined groups.
Since there were no significant differences found neither in the overall abortion frequency among three abortifacient bacteria nor occurring among naïve, primiparous and multiparous goats and sheep. These results agree with the findings of Benkirane et al. (2015). Tekle (2016) have reported that parity and abortion frequency have no significant effect on the Brucella infection. Gul et al. (2015) in Punjab, Pakistan have noticed higher prevalence of brucellosis in multiparous animals (6-10 parity) as compared to nulliparous (naïve animals), statistically significant (P< 0.004) through Rose Bengal Plate Test, but non-significant through Indirect ELISA and all other serological tests. However, our findings are not in agreement with the finding of Hadush et al. (2013) who have observed effect of parity on prevalence of abortifacient bacterial pathogens. This difference might be due to managemental practices, rearing conditions and due to sampling methodology and timings. The present investigation was in accordance with the work of Kelkay et al. (2017) who have revealed that there was no significant effect of parity and pregnancy status on abortion.
The isolation of Brucella melitensis from the vaginal material and milk is also of great public health importance (Habtamu et al., 2015). In our study among the total isolates one isolate from vaginal swab and four isolates from milk were recovered. However, no isolates were detected from foetal membrane. This could be due to the slow growing nature of the bacterial pathogens and rapidly growing contaminants (Seleem et al., 2010). The current, 12.5% B. melitensis isolates from serologically positive animals with previous record of abortion is in correlation with preceding studies of B. melitensis (Cekovska et al., 2010). A researcher from Ethiopia has reported 13.84% B. melitensis isolates from milk and vaginal swab samples of aborted goats (Tekle, 2016). Among all isolates, 4 (12.12%) isolated B. melitensis from milk are parallel to the different previous findings, 3.2%, Brucella were isolated from cattle milk in Pakistan (Ali et al., 2014) and 4.4% in Turkey (Celebi and Otlu, 2011).
The bacterial species B. melitensis was susceptible to tetracycline, doxycycline and ciprofloxacin whereas resistant to gentamycin, erythromycin, streptomycin, cefoxitin, lincomycin, bacitracin, rifampicin and cephalothin. Almost identical results regarding the sensitivity of B. melitensis to oxytetracycline and other drugs were noted by Ilhan et al. (2013) who found B. melitensis highly sensitive to tetracycline.
CONCLUSION
It is concluded that the abortifacient bacteria Chlamydia abortus, Coxiella burnetii and Brucella melitensis are prevailing in goats and sheep of district Dadu, Sindh. The present study confirms a considerably high prevalence of C. abortus and C. burnetii in sheep and goats. The parity and abortion frequency had no significant effect on the seropositivity of Coxiella burnetii, Chlamydia abortus and Brucella melitensis. Brucella melitensis was found resistant against the antibiotics erythromycin, lincomycin, rifampicin, cephalothin, bacitracin, streptomycin and gentamycin.
Funding
This research was funded by Sindh Agricultural Growth Program ‘Competitive Agricultural Research Development Fund’ (SAGP-CARDF). The study also acknowledged the CVDL, Tandojam for support in this research.
IRB aproval
The study was approved by the IRB, Directorate of Advanced Studies, SAU, Tnadojam (No. DAS/2674 of 2019).
Statement of conflict of interest
The authors have declared no conflict of interest.
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