Introduction
Coxiella burnetii (C. burnetii) is an obligate intracellular bacteria that causes Q fever, a zoonotic illness [1]. It is a tiny, pleomorphic Gram-negative bacteria that grows rapidly within the phagolysosome of eukaryotic phagocytes, with an estimated doubling time of 12–20 hours [2]. Despite being historically thought of as a Rickettsia, DNA sequence research places the Coxiella genus in the same family as Aquicella and Rickettsiella, the Legionellale order [3]. With the possible exception of Antarctica and New Zealand where its existence has not been established as of 2003, the disease has been known since the 1930s and is present around the world [4].
Globally, there is a growing interest in Q fever even in nations where the disease is thought to be extremely rare [5]. The disease is considered a re-emerging zoonosis in numerous countries [1]. Numerous animal species, including birds, mammals, and arthropods, are susceptible to infection by C. burnetti. The most common reservoirs of human infection are cattle, sheep, and goats [5].
In livestock, C. burnetii infection leads largely to reproductive problems such as miscarriages, stillbirths, poor calf delivery, metritis, and infertility with an associated economic burden on the herd [1]. Animals that shed C. burnetii mostly through the placenta and birth fluids, although ruminants can also expel the bacteria through their vaginal mucus, milk, feces, urine, and semen [6].
Humans with Q fever may experience an acute sickness (such as a self-limited febrile illness, pneumonia, or hepatitis) or a chronic illness (such as endocarditis in immunocompromised people, valvulopathy, miscarriages, or stillbirths in pregnant women) [7]. The primary method of human infection is inhaling contaminated dust or aerosols carried by diseased animals that contain C. burnetii [8].
Ticks are thought to be a key reservoir and vector of C. burnetii in various countries and more than 40 tick species are susceptible to C. burnetii infection [9].
Studies have also shown the prevalence of Q fever in different countries using different diagnostic methods [1].
There have been 18 documented cases of Q fever between 1999 and 2004 involving 289–299 individuals from 12 different countries [10].
With 2,257 human cases reported in the Netherlands in 2010, Q fever has emerged as a public health concern since 2007 [11]. Several studies have reported different prevalence rates of the infection in different parts of the world, signifying its importance. There have also been reports of prevalence rates in a few African countries with different prevalence rates reported.
Using the Card Agglutination test, it was discovered that 11% of cattle, 16.5% of sheep, and 8.8% of goats in Nigeria’s abattoirs in Kaduna and Zaria were seropositive [12]. In Kano, Sokoto, and Zaria slaughterhouses, C. burnetti was found in 12% of single-humped tropical camels [13].
Using indirect enzyme-linked immunosorbent assay (ELISA), [14] identified an 11.4% seroprevalence in sheep from Yobe state, whereas a 14.5% seroprevalence was reported in cattle in Kaduna, Kaduna state [15].
Without a recognized health status, food animals are brought into and out of Nigeria without being checked for infections. This is done for commerce and grazing purposes, and the animals could potentially infect people and other vulnerable animals at markets and slaughterhouses.
Meat and dairy consumption are common among the research area’s population, and animal husbandry practices that involve primarily cohabiting animals and humans in the same habitat offer a serious concern to public health since they can facilitate effective contact and organism spread. Presently, only a few studies exist in some Nigerian states on the current burden of C. burnetii in animals and humans, with the majority carried out in the 70s–80s [12,16].
Thus, this study attempted to detect C. burnetii infection in cattle slaughtered at the main abattoir in Sokoto because there is no available information on the infection/disease in ruminants from the study area.
Materials and Methods
Study area
Situated in the far northwest of Nigeria, at 13°05’N and 05°15’E, is the geographic coordinate of Sokoto. It has a total size of about 25,973 square km. The last national census in 2006 reported the state human population to be 3,702, 676 with annual rainfall between 500 and 1,300 mm and humidity range from 10% to 90% [17]. The state has abundant animal resources, including an estimated 3 million sheep, 4,600 camels, 5 million goats, 3 million cattle, and a variety of other domestic and foreign poultry species [18].
The study took place in the main abattoir in Sokoto, which is situated in the neighborhood known as ́kasuwan daji ́, off the western by-pass road in the vicinity of latitude 13°3’10.662” N and longitude 5°13’30.306” E, in Sokoto north local government. The abattoir is situated on a highland, which makes it easy for the effluents to drain directly into surrounding farmlands [19] Figure 1.
Study design
A cross-sectional study (for 6 months) and a systematic random sampling technique were employed. Every fifth cattle that enters the slaughter hall was selected for investigation [20]. Cattle of all ages, sexes, and breeds presented for slaughter were considered for sampling.
Information on the sex, age, and breed of animals sampled and the presence or absence of ectoparasites were recorded on a data form.
Sample collection, transportation, and storage
Five ml (5 ml) of blood sample each was collected from cattle immediately after slaughter and transported in an ice pack to the Central Research Laboratory, Faculty of Veterinary Medicine, Usmanu Danfodiyo University, Sokoto, where the sera were kept at –20°C until analysis.
Serological analysis
An indirect ELISA FQS-MS-5P kit obtained from ID.vet Innovative Diagnostics Grabels, France was used for this study. Briefly, microtiter wells were coated with C. burnetii phase I and II strain antigens. Test samples and controls were added to the wells. Anti-C. burnetii antibodies present formed an antigen-antibody complex. An anti-multi-species horseradish peroxidase (HRP) conjugate was added to the wells and fixed to the antibodies, forming an antigen-antibody conjugate–HRP complex. After the elimination of excess conjugate by washing, substrate solution (TMB) was added.
A blue solution appears that becomes yellow after the addition of the stop solution in the presence of antibodies and no coloration was observed in the absence of antibodies.
The microplates were read at 450 nm using a plate reader (JP select eng 2 2015).
Molecular analysis
Genomic DNA extraction from the sera of ELISA-positive samples
DNA was extracted from sera samples using a genomic DNA extraction kit according to the manufacturer’s instructions (QIAGEN, Hilden, Germany).
Determination of DNA quality and concentration
The concentration (ng/ µl) and purity of the extracted DNA were analyzed using an Eppendorf Biophotometer Plus (Eppendorf, Germany) and a NanoDrop® ND-1000 UV-VIS spectrophotometer (ThermoFisher Scientific, Wilmington, USA). The purity of DNA samples with an absorbance ratio at A260/A280 of 1.8–2.0 was only used in DNA amplification using polymerase chain reaction (PCR). DNA samples were alquited (10 µl) in tubes and stored in a −20°C freezer until required.
PCR cocktail for amplification of the C. burnetii genome
A total volume of 25 µl PCR reaction mix contained 12.5 µl of Mastermix (Biolabs®), 1 µl of Trans 1 (5’TATGTATCCACCGTAGCCAGTC-3’), 1 µl of Trans 2 (5’-CCCAACAACACCTCCTTATTC-3), 6.5 µl of nuclease energy-free water (Biolabs®) and 4 µl of DNA template.
The expected amplification product was 687-bp.
PCR protocol
A modified PCR protocol was adopted [21]. The tubes were transferred into an applied biosystem 9,700 thermocycler programed with the following conditions: initial denaturation at 95°C for 30s, followed by 40 consecutive cycles of denaturation at 95°C for 30s annealing at 95°C for 30s, extension at 60°C for 30s, extension at 72°C for 1 minute and a final extension at 72°C for 7 minutes.
Gel electrophoresis of the PCR products
The PCR products were loaded in 1% agarose gel (Vivantis Incorp, USA) and 1 TBE (Vivantis Incorp, USA) buffer (Tris 0.09M- borate 0.09M—EDTA 0.02M) stained with ethidium bromide (Biotium, Hayward, USA). Electrophoresis was performed at 70 V for 55 minutes (Thermo Owl Separation Systems, USA). Molecular markers of 100 bp (GeneDirex, Taiwan) were run in parallel with the DNA samples as an indicator of the size of the PCR amplicon. Gels were visualized using a UV light transilluminator (Major Science, USA) and photographed.
Data analysis
The data obtained from this study were subjected to descriptive and inferential statistics (chi-square test and simple logistic regression) to determine the association of the variables (age, sex, breed, and presence or absence of ectoparasites) with the presence of C. burnetii antibodies.
A value of p < 0.05 was considered significant in the studies, and statistical software ‘SPSS’ version 22.0 was used for the statistical analysis
Results
A total of 184 sera samples were collected from the main abattoir in Sokoto and screened for the presence of C. burnetii antibodies using an indirect ELISA kit from Idvet®. Of these, 10 were positive, constituting an overall prevalence of 5.4% (Table 1).
Table 1.
Overall seroprevalence of C. burnetii infection in Cattle slaughtered at Sokoto main abattoir.
| Sex | Number of sample | Positive | Prevalence (%) |
|---|---|---|---|
| Male | 121 | 4 | 2.2 |
| Female | 63 | 6 | 3.2 |
| Total | 184 | 10 | 5.4 |
Table 2 shows the distribution of C. burnetii antibodies based on sex. The sex-specific prevalence revealed that cows had a higher number of positive samples of 6 (9.5%), whereas bulls cattle had a lower prevalence of 4 (3.3%). The differences between the sexes were not significant at the 5% level of significance as the p-value was greater than 0.05 (0.094).
Table 2.
Univariable analysis (Fisher’s Exact) showing the relationship between C. burnetii antibodies and sex in cattle slaughtered at Sokoto main abattoir.
| Variable | Level | Prevalence (%) | p-value | OR | 95%CI |
|---|---|---|---|---|---|
| Sex | Male | 4/184 (2.17) | Ref | Ref | NA |
| Female | 6/184 (3.26) | 0.094 | 3.079 | 0.836–11.345 |
P=0.094, OR=3.079, 95% CI=0.836–11.345.
OR, Odds ratio; ref, reference category; NA, not applicable; CI, Confidence interval.
The age-specific prevalence of C. burnetii antibodies revealed that the positive samples were from adult cattle at 6.7%. Fisher’s exact test was not significant at the 5% level of significance (p=0.213), Table 3.
Table 3.
Univariable analysis showing the relationship between C. burnetii antibodies and age.
| Variable | Level | Prevalence (%) | p-value | OR | 95%CI |
|---|---|---|---|---|---|
| Age | Young | 0/35(0%) | Ref | Ref | NA |
| Adult | 10/149(6.7) | 0.213 | NA | 1.027–1.119 |
P=0.213, OR=1.072, 95% CI=1.072–1.119.
OR, Odds ratio; ref, reference category; NA, not applicable; CI, Confidence interval.
Table 4 shows the distribution of C. burnetti antibodies based on tick presence or absence. The study revealed that cattle with ticks had a higher seroprevalence (27.8%) than those without ticks (3.0%). A statistically significant association was found between the presence of ticks and C. burnetii antibodies at the 5% level of significance (p=0.001).
Table 4.
Relationship between C. burnetii antibodies and presence of ticks.
| Variable | Level | Prevalence (%) | p-value | OR | 95%CI |
|---|---|---|---|---|---|
| Ticks | Absent | 5/166(3.0) | Ref | Ref | NA |
| Present | 5/18(27.8) | 0.001 | 12.385 | 3.171–48.365 |
χ2=19.38, p=0.000, 0R=12.385, 95% CI=3.171–48.365.
OR, Odds ratio; ref, reference category; NA, not applicable; CI, Confidence interval.
The breed-specific prevalence of C. burnetii antibodies showed that White Fulani had the highest number of positive samples of 5 with a prevalence of 6.5%. In contrast, Sokoto Gudali had the lowest number of 2, with a prevalence of 3.4%, as shown in table 5.
Table 5.
Logistic regression analysis showing the relationship of C. burnetii antibodies with breeds of cattle.
| Variable | Level | Prevalence (%) | p-value | OR | 95%CI |
|---|---|---|---|---|---|
| Breed | Red Bororo | 3/49(6.1) | 0.519 | 0.548 | 0.088–3.418 |
| Sokoto Gudali | 2/58(3.4) | Ref | NA | NA | |
| White Fulani | 5/77(6.5) | 0.934 | 1.065 | 0.243-4.670 |
OR, Odds ratio; ref, reference category; NA, not applicable; CI, Confidence interval.
The ten seropositive samples from ELISA were subjected to molecular analysis. However, no amplicon was observed on 1% agarose gel after amplification of the internal transposon region of the C. burnetii genome using polymerase chain reaction (PCR).
Discussion
Q fever is an emerging disease with the potential to spread and become endemic in new regions of the world. The risk of this disease is elevated because it is capable of using several host and arthropod species as reservoirs and vectors for transmission, respectively. Infected cattle are important reservoirs of the disease and are a major source of infection in humans [22].
The current seroprevalence of the disease in the study area is lacking, and there is a dearth of information on the disease in Nigeria. Currently, there is a reported seroprevalence of 14.5% in intensively reared cattle in Kaduna [15], and 11.7% in sheep from Yobe state, Nigeria [14]. Seropositivity to C. burnetti in cattle (ranging from 5.56% to 15.6%) was also obtained from Kwara, Plateau, and Borno in Nigeria [23].
However, in this study, the overall prevalence of C. burnetii antibodies was 5.4%. This is lower than the overall prevalence reported in Nigeria by [12,15], and [23] who reported a prevalence of 11% and 14.5%, 15.6%, 9.4%, and 5.56% in cattle, respectively. The overall prevalence is also lower than in other African countries and other parts of the world [24].
The differences observed in the overall prevalence could be attributed to variations in geographic location, period of the study, number of beef animals slaughtered compared to dairy, higher sensitivity and specificity of the test used, sample size, and technique used.
However, the prevalence obtained from this study is higher than the prevalence obtained by [25], who reported a prevalence of 4% in Chad. This difference could be attributed to the smaller number of samples used in the studies.
The sex-specific prevalence of the disease revealed that cows had a higher prevalence than male bulls. The higher prevalence in cows could be attributed to the fact that the organism has a higher affinity for the placenta, mammary gland, and fetal membranes. However, there is no significant statistical association, which agrees with the findings of [15] and [25].
The age-specific prevalence of the infection also revealed that adult cattle had all positive samples. However, there was no significant statistical association. This could be attributed to the fact that adult cattle are slaughtered more than young cattle and also due to a high possibility of contact with the organism with an increase in age. Significant association with age was not found by [15] and [25]. This, however, varies with another study that reported a significant association between adult cattle and heifers [26].
The breed-specific prevalence showed that White Fulani had a higher number of positive samples with a prevalence of 6.5%, whereas Sokoto Gudali had the lowest number of positive samples with a prevalence of 3.4%. There was no statistical association between breeds and the presence of antibodies. The relatively high prevalence observed in White Fulani could be due to the predominance of the breed slaughtered during the sampling and slaughter periods.
In this study, a significant statistical association was found between the presence of C. burnetii antibodies and the presence of ticks on the cattle sampled (p < 0.001). This could be due to the possibility that the animals presented for slaughter in the abattoir had no ectoparasite control and the significance of ticks in transmitting the disease in ruminants had been documented [27]. The findings of this study agree with the work of [28], who reported the presence of ticks on animals as a risk factor associated with C. burnetii infection. However, the findings vary with the work of [29], who reported no statistical association between seropositivity and the presence of ticks in cattle.
No active infection was detected in seropositive sera by PCR. The diagnosis of Q fever is made by serological tests, the culture of the organism, and conventional PCR. The negative result obtained using PCR observed in this study may be due to seroconversions, which normally occur 3–4 weeks after infection. Antibodies against C. burnetti often appear late during the disease, which makes it difficult to diagnose in the early stage. DNA-based methods are said to be the best method for early diagnosis, and methods such as PCR, nested PCR, and real-time PCR have been successfully used for the clinical identification of C. burnetii [30].
The finding is similar to the work of [31], who had a seroprevalence of 6.97% using an antibody ELISA kit but obtained no prevalence using RT-PCR. It also agrees with the findings of [32], who reported positive values in ELISA-tested sera samples, but all real-time PCR results were negative. However, the findings from this study do not agree with the work of [33], who obtained high seropositivity from ELISA (10.5%) and 1.5% prevalence from PCR analysis.
Conclusion
The overall seroprevalence of Q fever obtained was 5.4%, signifying that some cattle slaughtered at the main abattoir in Sokoto were C. burnetii infected, and seropositive samples subjected to PCR did not reveal active C. burnetii infection in the study.
The variable of the presence of ticks appears to be a risk factor for C. burnetii infection in this study.
This study is important because it highlights the presence of C. burnetii in cattle in Sokoto, identifies ticks as a risk factor, and recommends further research and control measures. These findings are essential for improving animal and public health in the study area.
It was recommended that further studies should be conducted to determine the prevalence of the disease and detect C. burnetii in breeding herds and on other species of ruminants such as sheep and goats because they are reared together in the study area.
Ectoparasites, especially ticks, should be closely studied to determine their infection rate and role in disease transmission in the study area.
List of Abbreviations
C. burnetii, Coxiella burnetii; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction; OR, odds ratio; Ref, reference category; NA, not applicable; CI, Confidence interval.
