Serological monitoring and risk factors of brucellosis and Q fever in calves in Türkiye
Article information
Abstract
The death and loss of offspring before the gestation period is complete is defined as abortion. All calf losses before the 200th day of pregnancy are defined in this way. Brucellosis and Q fever diseases are among the most important abortive diseases in cattle. This study examined the seropositivity rates of brucellosis and Q fever in 3- to 6-month-old calves not vaccinated with Brucella abortus S19. Six hundred and 81 calves were included, and blood serum samples were collected. The rose Bengal plate test (RBPT), indirect enzyme-linked immunosorbent assay (ELISA), and complement fixation test (CFT) tests were used to diagnose brucellosis, and indirect ELISA was used to diagnose Q fever. Among the calves whose blood serum was collected, the positive results for the RBPT, indirect ELISA, and CFT tests were 3.5%, 8.8%, and 5.5%, respectively. A positivity rate of 6.6% was determined for Q fever. In the study, the co-infection rate of brucellosis and Q fever was determined to be 0.44%. On the other hand, the seropositivity of brucellosis and Q fever in calves was not significant (p > 0.05) according to sex and age. The calves born in the study area were infected either intrauterine or during the postpartum period. In addition, ELISA had higher sensitivity than the other tests. Therefore, the combination of RBPT and indirect ELISA should be used in herd screenings to detect more infected animals.
Introduction
Abortion is defined as the death and loss of the fetus before the completion of the gestation period. In cattle, all fetal losses occurring before the 200th day of gestation are classified as abortion. The causes of fetal losses can be categorized into infectious and non-infectious factors. Approximately 10% of abortions in cattle are attributed to non-infectious causes [1]. Infectious abortions are caused by bacteria, viruses, fungi, and protozoa [1–4] and can occur endemically or sporadically within herds [2].
Brucellosis is a zoonotic disease characterized by abortions, commonly caused by Brucella species in animals. The disease can be observed in various domestic and wild animal species, depending on the pathogen species. Bovine brucellosis is prevalent in many countries. The disease exhibits an asymptomatic course in young and non-pregnant animals. Even when abortions are not observed in infected pregnant animals, the bacteria are shed into the environment through fetal fluids, vaginal discharges, milk, and placentas [5]. In bovine brucellosis, although abortion cases are encountered, normal calf birth can be observed. In such cases, the calf can be infected vertically while in the uterus or through milk after birth [6,7]. Rhyan et al. [6] conducted serological examinations of calves born to seropositive and seronegative dam animals. While all calves born to seronegative dams tested negative, 71% (5/7) of calves born to seropositive dams were seropositive [6]. Approximately 60% to 70% of fetuses born to brucellosis-positive dam animals are born infected because of vertical transmission, posing a significant risk for control [8]. In bovine brucellosis, after bacteria transmission, subsequent pregnancies may result in abortions. Although 80% of infected cattle do not experience a second abortion, the spread of the bacteria and the infection within the herd can occur [8].
Q fever is an important zoonotic disease caused by Coxiella burnetii, an obligate intracellular pathogen. The disease is generally characterized by abortions in ruminants and can cause respiratory and cardiovascular system disorders in humans. The disease is ubiquitous worldwide except in New Zealand. C. burnetii is shed into the environment by infected animals through vaginal discharges, feces, and milk, serving as a source of transmission to other animals. Ticks also play a role in transmission. The primary transmission source to humans is milk and dairy products [9]. Wind can also contribute to transmission from contaminated areas [10]. Serological or molecular testing methods are used for routine disease diagnoses [9]. Among the serological methods, enzyme-linked immunosorbent assay (ELISA) has higher sensitivity and specificity than the complement fixation test (CFT) [9,10].
Maternal antibodies are a favorable condition for protecting calves against Q fever, but they can suppress the development of active immunity after vaccination. Therefore, it is preferable to administer vaccines during a period of low maternal antibody levels to establish effective immunity in calves [11]. Despite the numerous studies on brucellosis and Q fever in adult cattle worldwide, studies specifically on the prevalence in calves are limited. Understanding the situation in calves will provide important data for the epidemiology of these diseases and their spread within herds. Vaccination before the infectious agent is transmitted is necessary for the vaccine to be protective against many diseases, especially brucellosis. Therefore, for vaccination against brucellosis to be successful, calves should not be infected intrauterine or after birth before vaccination. This study examined the prevalence of brucellosis and Q fever diseases in calves in Ardahan, Erzurum, and Kars provinces, where cattle breeding is intensively carried out in Türkiye. In addition, the effects of various factors on these diseases were investigated.
Materials and Methods
Ethics statements
This research was conducted at the Erzurum Veterinary Control Institute under the Ministry of Agriculture and Forestry, with the support of the General Directorate of Agricultural Research and Policies. Ethical approval for collecting blood samples from calves was obtained from the Local Ethics Committee for Animal Experiments of the Erzurum Veterinary Control Institute (approval number: 13067196/122).
Sample size and sampling
The study was conducted in at least three districts in Ardahan, Erzurum, and Kars provinces, which are within the working area of the Erzurum Veterinary Control Institute. In Ardahan, Erzurum, and Kars provinces, calf births are generally observed in February–March. Therefore, blood sera were collected from calves aged 3 to 6 months between June and August for blood sampling. The estimated herd and individual prevalence in the study area were assumed to be 10% and approximately 15%, respectively [12]. Sample size calculations were conducted to estimate the prevalence. For this purpose, the number of cattle farms and the number of calves that should be sampled in each province were calculated. Previous Ministry of Agriculture and Forestry survey results were used to estimate the prevalence. Considering a 95% confidence interval, a 5% margin of error, and an assumed prevalence of 10%, it was determined that samples should be collected from at least 139 farms. Considering a 95% confidence interval, a 5% error, and an assumed prevalence of 15%, samples should be collected from at least 196 calves. The districts where cattle breeding is intensive were determined during sampling.
Sample collection
Ardahan, Erzurum, and Kars are provinces where pasture-based livestock farming is prevalent, and the herds from which blood samples were collected are those engaged in pasture-based livestock farming. Blood samples were taken from calves aged 3 to 6 months old that had not been vaccinated with the Brucella abortus S19 vaccine. All calves sampled were taken from the crossbred Brown Swiss breed, which is raised widely in the region. In this study, the calves from which blood would be taken were determined randomly, and no sex discrimination was made. Samples were taken from healthy-looking calves on the farms. No samples were taken from animals showing disease symptoms, such as diarrhea or pneumonia. Information on the sex, age, ear tag number, and farm number was obtained for the animals from which the blood serum samples had been collected. The collected blood sera were transported to the laboratory at 4°C. The serum was separated from blood samples by centrifugation at 3,000 rpm for three minutes. Clear sera without hemolysis were considered suitable for analysis. The separated sera were placed in 2 mL microcentrifuge tubes. Serum samples were stored at −20°C until the analyses were performed.
Brucellosis serological tests
Indirect ELISA and rose Bengal plate test (RBPT) with the smooth rose Bengal plate antigen were performed to diagnose Brucella spp.-specific immunoglobulin (Ig) G and IgM in the serum samples [5]. For serum samples detected as positive in indirect ELISA (Innovative Diagnostics, France) and RBPT (Dollvet, Türkiye) analyses, a CFT was performed for titer determination. National anti-Brucella standard antiserum was used as a positive control in each RBPT and CFT analysis.
The calf sera taken in the study were analyzed by indirect ELISA according to the manufacturer’s protocol. The RBPT, used for brucellosis diagnosis from calf sera, was conducted in an accredited laboratory within the scope of ISO/IEC 17025:2017. A 50 mL serum sample was transferred onto the RBPT analysis plate. An equal amount of smooth Brucella antigen was added to the serum and mixed. The presence of agglutination was examined in the resulting circle, approximately 2 cm in diameter [5].
CFT was carried out in an accredited laboratory within the scope of ISO/IEC 17025:2017. All sera were diluted at a ratio of 1:5, and the positive control was diluted to 1:20 to observe a 1/1,280 dilution of the serum. The prepared plates were incubated overnight in a refrigerator, and a 2% hemolytic system was added. After adding the hemolytic system, incubation was carried out at 37°C for 30 minutes. After incubation, the samples were centrifuged at 1,500 rpm for five minutes, and the test was evaluated according to the standard protocol [5].
Q fever serology
For the serological examination of Q fever, the indirect ELISA (Innovative Diagnostics) method was used to diagnose phase I and phase II C. burnetii -specific IgG, which is indicated by the World Organization for Animal Health (WOAH) as the serological test method with the highest sensitivity and specificity. A commercial ELISA kit was used for the test [9]. The calf sera taken in the study were analyzed by indirect ELISA according to the manufacturer’s protocol.
Statistical analysis
The age and sex data of the calves sampled in the study were compared with brucellosis and Q fever positivity. The distribution of brucellosis and Q fever positivity according to age and sex was evaluated for significance using the Wilcoxon test, and p-values < 0.05 were considered significant [13].
Results
In the research conducted to determine the status of brucellosis and Q fever in calves, 681 blood serum samples were collected from Ardahan, Erzurum, and Kars provinces (Fig. 1). Table 1 lists the distribution of blood serum samples according to provinces.
View of the provinces of Ardahan, Erzurum, and Kars, where the research was conducted on the map. (A) Locations and borders of Ardahan, Erzurum, and Kars provinces on the map. (B) Appearance of the number of positive samples at the district level on the heat map according to the brucellosis serological examination. The highest positivity rate was detected in Ardahan Göle district. (C) Appearance of the number of positive samples at the district level on the heat map according to the Q fever serological examination. The highest positivity rate was detected in Kars Sarıkamış district. Scale bars: (B) 0-9, (C) 0-13.
Distribution of blood serum samples taken according to the provinces and districts and distributions of brucellosis and Q fever positivity
During the study period, 681 blood samples were collected from calves, and brucellosis positivity was detected in 5.6% (38/681) of the calves. RBPT, indirect ELISA, and CFT analyses were performed separately for brucellosis analysis. The percentages from these analyses were 3.5%, 8.8%, and 5.6%, respectively (Fig. 2). The indirect ELISA method was used for the analysis of Q fever, and specific antibodies against C. burnetii were detected in 6.6% (45/681) of the samples.
Showing the positivity according to the tests used to analyze brucellosis in the column chart. RBPT, rose Bengal plate test; CFT, complement fixation test; indirect ELISA, indirect enzyme-linked immunosorbent assay.
In the study, the co-infection rate of brucellosis and Q fever in the 3- to 6-month-old calves was 0.44% (Table 2). An analysis of the connection between disease positivity and age revealed three months of age to be predominant (Fig. 3). Nevertheless, the seropositivity of brucellosis and Q fever in calves was not significant (p > 0.05) according to sex and age (Table 3).
Number of co-infections of brucellosis and Q fever diseases in calves
Box chart showing the distribution of positivity in brucellosis and Q fever diseases according to age.
Statistical evaluation of the distribution of brucellosis and Q fever diseases in calves by age and sex
Discussion
Brucellosis is a disease caused by Brucella spp. agents and manifests as abortion in cattle [5]. Despite eradication efforts in many countries, the disease is still prevalent on all continents globally, considering both wild and domestic animal populations [14]. Q fever, which is caused by the intracellular pathogen C. burnetii, causes abortions in cattle. Q fever is present in all countries except New Zealand [10,15]. Brucellosis and Q fever can be transmitted vertically from the dam to the offspring, leading to persistent infection [16,17]. Maternal antibodies do not pass to the offspring, owing to the epitheliochorial placental structure in cattle. Therefore, the absence of specific antibodies against an antigen in precolostral blood samples taken from calves indicates that the calf has not encountered the intrauterine antigen or has developed tolerance to the antigen [18]. The seroprevalence of brucellosis and Q fever infections in calves without maternal antibodies was determined by analyzing the blood samples of 681 calves aged 3 to 6 months from Ardahan, Erzurum, and Kars provinces, where the B. abortus S19 vaccine was not administered.
Brucellosis can also be transmitted vertically from the mother to the offspring [16]. Plommet et al. [19] reported a vertical transmission rate between 60% and 70%. Vertical transmission can occur intrauterine and during passage through the birth canal. Moreover, calves can become infected during the colostrum and suckling period. These infected calves pose the greatest threat to eradication programs. Infected cattle usually abort in their first pregnancies, and 80% of them do not abort in subsequent pregnancies. Nevertheless, they shed the microorganism through birth discharges and milk [8]. Fernández et al. [20] examined the serum samples from 192 calves in their research conducted in Mexico and found positivity in 23% of the calves [20]. Merga Sima et al. [21] conducted a seroprevalence study in Ethiopia and reported a brucellosis seroprevalence rate of 0.8% in six-month-old calves. The simulations used in brucellosis eradication strategies accept a 20% rate of vertical transmission in cattle [22]. Yahyaoui Azami et al. [23], in their research conducted in Morocco, detected a seroprevalence of 13.7% in animals under one year of age. Few studies have examined the prevalence of brucellosis in calves and vertical transmission. The brucellosis seropositivity rate obtained in this study is consistent with other research findings.
Freick et al. [24] conducted a study in Germany, which is endemic to Q fever. They examined precolostral serum samples from 56 stillborn calves and 30 live-born calves of infected cows. A positivity of 7.1% was detected in stillborn calves, while no positivity was found in live-born calves. The absence of antibodies in live-born calves was attributed to the insufficient sample number [24]. Literák and Calvo Rodríguez [25] conducted a serological examination of Q fever in cattle in the Czech Republic. They observed seropositivity in 2.8% of calves in two out of three farms and 14.4% in calves in one farm [25]. Tutusaus et al. [18] conducted a study examining seropositive and seronegative cows’ calves for serological examination of Q fever from the precolostral period onwards. The researchers did not detect antibodies in the calves of seronegative and seropositive animals during the precolostral period [18]. Radinović et al. [17] identified C. burnetii DNA through polymerase chain reaction in the blood of six healthy calves born from nine seropositive cows, confirming intrauterine transmission of the pathogen. Tutusaus et al. [18] evaluated inactive vaccination. They reported that specific maternal antibodies against Q fever could be detected in the blood serum of seropositive and vaccinated adult cows’ calves from colostrum up to three months after birth. Similar studies reported seropositivity rates in calves ranging from 0% to 14.4%. The seropositivity rate found in this study is consistent with previous research.
Regarding the serological examination of brucellosis, the positivity rates using RBPT, indirect ELISA, and CFT analysis were 3.5%, 8.8%, and 5.5%, respectively. Khan et al. [26] examined cattle seroprevalence in Pakistan. They reported seropositivity rates of 12.0% with RBPT and 18.5% with indirect ELISA in blood serum samples. RBPT and indirect ELISA are screening tests. After these tests, quantitative tests should be used to measure the antibody titers and evaluate the results [5]. CFT, however, is a confirmatory test accepted for international trade [27]. In this study, the highest positivity rate was detected with indirect ELISA. Erdenliğ Gürbilek et al. [28] compared RBPT, CFT, and indirect ELISA tests to analyze cattle blood serum samples for bovine brucellosis. They reported that indirect ELISA had the highest seropositivity rate. ELISA is considered the most sensitive test in extensive serological screenings for bovine brucellosis. It also enables the early detection of latent infections [29]. ELISA and RBPT, being highly sensitive tests, tend to have higher positivity rates than CFT because screening tests are more prone to false positive reactions due to their higher sensitivity [30]. This is because RBPT and ELISA tests detect both IgG and IgM antibody isotypes, while CFT only detects the IgG1 isotypes [31]. Most false positive reactions in serological diagnosis of brucellosis are actually due to IgM isotypes. Therefore, the CFT test is considered more specific [31]. Kashiwazaki et al. [32] reported that combining RBPT and indirect ELISA tests to screen infected herds is the most effective method for detecting infected animals.
Khan et al. [26] reported a seropositivity rate of 4.7% and 27.1% in young and adult animals in Pakistan, with a positive correlation between age and seropositivity. Boukary et al. [33] examined the risk factors of brucellosis in cattle, sheep, and goats in Niger. They reported a seropositivity rate of 1.8% in calves under one year of age [33]. The spread of Q fever in cattle was more effective through horizontal than vertical transmission [34]. Wainaina et al. [35] examined the seroprevalence of Q fever in cattle and found a seroprevalence rate of 8.2% in young cattle. Aljafar et al. [36] reported a seroprevalence rate of 1.0% in cattle under one year of age. Age is a significant risk factor for seropositivity in Q fever. This phenomenon can be explained by the increased rate of exposure with age [37]. No research has examined the distribution of brucellosis and Q fever seropositivity in calves according to monthly ages. The present study showed no significant difference in seropositivity rates among age groups.
Yahyaoui Azami et al. [23] reported seropositivity rates for brucellosis of 22.7% in females and 12.0% in males, but no significant sex differences were found. Similarly, Khan et al. [26] found rates of 25.4% and 6.5% in females and males, respectively, also without statistical significance. Boukary et al. [33] observed similar seropositivity rates in males (3.3%) and females (3.2%). In Nigeria, Cadmus et al. [38] detected a higher brucellosis positivity in females (12.8%) than in males (4.7%). For Q fever, Ibrahim et al. [39] reported a 10% seropositivity in female animals and 1% in males in Ethiopia. McCaughey et al. [34] observed a 6.3% and 1.1%seroprevalence in female and male cattle in Northern Ireland, respectively. In Kenya, Wainaina et al. [35] recorded a Q fever prevalence of 24.2% and 7.8% in females and males, respectively, while Aljafar et al. [36] reported rates of 17.7% and 0.8% in females and males, respectively in Saudi Arabia. Across these studies, although the seropositivity rates varied between sexs, the differences were not significant (p > 0.05), aligning with other similar research findings [23,26,33-36,38,39].
Troupin et al. [40] conducted a seroprevalence study on brucellosis and Q fever in Guinea between 2017 and 2019 and determined a co-infection rate of 1.94% in cattle. Cadmus et al. [38] detected a Q fever and brucellosis co-infection rate of 0.87% in cattle. Hence, the mixed infection status of brucellosis and Q fever was very low. The co-infection rate was determined to be 0.44%. The findings of the presented research were consistent with similar studies.
This study had several limitations. Antibodies were used for diagnosis rather than the direct agents regarding intrauterine or postnatal transmission. The kind of clinical findings the infected animals showed regarding diseases could not be monitored after sexual maturity. In addition, the samples were not taken from the mothers of the sampled calves. The strength of the study is that the region where the research was conducted in Türkiye is the region where animal husbandry and animal movements are intense. No similar research has been conducted in Türkiye, meaning no comparison can be made on the effect of changes in brucellosis control strategies.
In summary, the prevalence of brucellosis and Q fever in calves was determined to be 5.5% and 6.6%, respectively. Mass vaccination of calves aged 3 to 6 months is the commonly applied method for combating brucellosis worldwide. This study was conducted on animals not vaccinated with the B. abortus S19 vaccine to determine the prevalence of infection in this age group based solely on infection-related antibodies. Ensuring calves are vaccinated before becoming infected is key to successful brucellosis control. According to the findings, it is necessary to consider that 5.5% of calves could remain reactor animals in the herd even after being vaccinated if they had been infected before vaccination. Cows that had previously aborted young due to brucellosis or Q fever may become reactor animals in farms and contribute to the spread of these diseases in later periods. Therefore, it is necessary to identify reactor animals on farms, report all abortion cases, and vaccinate all calves. Nevertheless, further research is needed to obtain epidemiological information on intrauterine or postpartum transmission of these diseases to calves.
The WOAH reports screening tests and confirmation tests for the diagnosis of brucellosis. Although high positivity was detected with indirect ELISA in the present study, some of the results from the CFT were false positive reactions. On the other hand, the combined use of indirect ELISA and RBPT in screening tests will provide a higher detection of infected animals within the herd. A comparison of the seropositivity of brucellosis and Q fever in calves with adult animals and an examination of the diagnosis of the agent from seropositive calves will provide information on the epidemiology of brucellosis and Q fever diseases.
Notes
Conflict of interest
The authors declare no conflict of interest.
Author’s Contributions
Conceptualization: Ozgen EK; Data curation: Ozgen EK; Formal analysis: Ozgen EK; Funding acquisition: Ozgen EK; Investigation: all authors; Methodology: Ozgen EK; Project administration: Ozgen EK; Resources: Ozgen EK; Software: Ozgen EK; Supervision: Ozgen EK; Validation: all authors; Visualization: all authors; Writing–original draft: Ozgen EK; Writing–review & editing: Ozgen EK, Sayi O, Atalay E.
Funding
This study was supported by the Republic of Türkiye Ministry of Agriculture and Forestry, General Directorate of Agricultural Research and Policies (project number: TAGEM/HSGYAD/B/21/A5/P1/2455, Türkiye).
Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.