Serological Evidence and Coexposure of Selected Infections among Livestock Slaughtered at Eastern Cape Abattoirs in South Africa

Zoonotic infections were investigated in a cross-sectional study on asymptomatic livestock slaughtered in abattoirs in the Eastern Cape. Antibodies against Brucella spp., Coxiella burnetii, Toxoplasma gondii, and the coexposure were investigated in sera using serological tests. A total of 565 animals comprising of 280 cattle, 200 sheep, and 85 pigs were screened using RBT, iELISA, CFT, and AMOS-PCR. The Mast® Toxoreagent test and iELISA were used for the detection of T. gondii and C. burnetii, respectively. The Brucella positivity based on at least two tests was 4.3% (12/280), 1.0% (2/200), and 0.0% (0/85) in cattle, sheep, and pigs, respectively. Toxoplasma gondii seropositivity of 37.90% (106/280), 1.50% (3/200), and 7.10% (6/85) was observed in cattle, sheep, and pigs, respectively. Coxiella burnetii seropositivity of 26.40% (74/280), 15.00% (30/200), and 2.40% (2/85) was observed in cattle, sheep, and pigs, respectively. Coexposure was detected in cattle for positivity against C. burnetii and T. gondii 40.54%, Brucella spp. and T. gondii 1.35%, and Brucella spp. and C. burnetii 4.05%. Coexposure for Brucella spp., C. burnetii, and T. gondii 4.05% was detected in cattle. Coexposure of Brucella spp. and C. burnetii 6.67% was detected in sheep. The AMOS-PCR identified B. abortus in cattle and a mixed infection of B. abortus and B. melitensis in sheep in 64.71% seropositive samples. To our knowledge, the coexposure of Brucella spp., T. gondii, and C. burnetii in cattle has not been reported. Coexposure of Brucella spp. and C. burnetii in cattle and sheep is significant as it results in reproductive losses and constitutes an infectious risk to humans. The detection of antibodies against multiple zoonotic infections in livestock from abattoirs has implications for public health.


Introduction
According to a study conducted by the Asian Pacifc Strategy for Emerging Diseases in 2010, approximately 60% of emerging human diseases are zoonotic and more than 70% of these pathogens come from wildlife species [1].It has been reported that over 14 million deaths are reported annually due to infectious diseases [2].Zoonotic diseases afect human and animal populations, resulting in illnesses, mortality, and decreased productivity [3].Tese diseases can have a signifcant social impact in endemic areas [3].Many pathogens causing abortion in animals may lead to severe human illness, particularly Toxoplasma gondii, Brucella spp., Chlamydophila spp., Campylobacter spp., Salmonella spp., Listeria spp., Leptospira spp., and Coxiella burnetii [4,5].In South Africa (SA), brucellosis, Rift Valley fever, and toxoplasmosis have been identifed as part of the seventeen diseases classifed as zoonotic priorities [6].Tere is currently a dearth of information on the incidence of these diseases in the human population of SA.Tis study was initiated to establish baseline data so that further studies can be undertaken for such high-risk populations.
Brucellosis and coxiellosis are highly contagious zoonotic infections of humans and domestic animals [7,8].In SA, Brucella abortus and B. melitensis mainly infect cattle and small ruminants (sheep and goats), respectively, and also infect humans while brucellosis has not been reported in pigs [9,10].Tese species have been reported in SA, frst in small ruminants and later in cattle as well as humans since the turn of the century [11][12][13], that later resulted in bovine brucellosis scheme to control brucellosis.Te scheme focuses on controlling B. abortus by vaccination of S19 and serological testing followed by seropositive slaughter in high-risk bovines such as dairy and export with voluntary participation for other livestock [14].WOAH recommends "unequivocal diagnosis of Brucella infections can be made only by the isolation and identifcation of Brucella," but in situations where bacteriological examination is not practicable, diagnosis must be based on molecular or immunological methods [10].WOAH recommends serological tests for the control of brucellosis at the national or local level such as Rose Bengal test (RBT), indirect ELISA (iELISA), complement fxation test (CFT), and fuorescent polarization assay (FPA) as suitable screening tests [10].Te diagnostic performance characteristics of ELISAs and FPA are comparable with or better than that of the CFT, and as they are technically simpler to perform and more robust, their use may be preferred [10].Te problem with SA partial brucellosis scheme focusing only on bovines is once the disease has been established in a herd, it is difcult to control as it has variable incubation periods ranging from several months to at least two years and even nine years as reported [15].Tese problems make control and elimination from herds costly and difcult as it takes a minimum of two years after removal of infected animals to declare a herd free and decades to declare countries free of brucellosis [15].As human brucellosis is mainly caused by contact with Brucellainfected animals that could be asymptomatic [16], their secretions and carcasses result in occupational public health risks for those workers in contact with animals such as abattoir workers, animal handlers, and veterinarians.Despite the endemic occurrence of brucellosis in SA, only a few reports in humans are available [17].
Coxiellosis in animals is caused by C. burnetii, which is a leading cause of abortions, decreased reproductive ability, and subclinical infections in ruminants [18].Coxiella burnetii has a biphasic development cycle.Te large-cell variant is a vegetative form found in infected cells, while the small-cell variant is the extracellular infectious form shed in milk, urine, vaginal secretions, semen, and faeces in both animals and humans.Te latter infectious form is also found in high concentrations in placental tissue and amniotic fuid [19], that persists for weeks to years in the environment [19].Similar to brucellosis, humans are infected with coxiellosis through infected animals which may be asymptomatic [20,21].Teir secretions result in occupational public health exposure potential for humans in contact with animals such as abattoir workers, animal handlers, and veterinarians.In SA, the frst human case of Q-fever was documented in 1950 [22] and C. burnetii has been reported in goats, cattle, and sheep [23,24] despite lack of surveillance programs.
Toxoplasma gondii is a ubiquitous protozoan parasite of warm-blooded animals [25], causing infections in humans and wild and domestic animals, including birds, cats, sheep, goats, cattle, and pigs [26].Despite the organism infecting humans, wildlife, and domestic animals, only members of the cat family (Felidae) have been confrmed as defnitive hosts for T. gondii infection.Unlike in humans, the clinical signs of toxoplasmosis infections in animals are nonspecifc [27,28], making diagnosis in animals by observation of symptoms insufcient.Te molecular and serological tests such as PCR, indirect hemagglutination assay, indirect fuorescent antibody assay, latex agglutination test, and ELISA tests are currently used [29].Serological results must be evaluated carefully because, according to bioassay investigations, most seropositive cattle lacked signs of active T. gondii infections [30].Despite the infection in cattle, abortions, and neonatal mortality are not common [31], in contrast, infection with T. gondii signifcantly contributes to abortion and stillbirth in sheep, goats, cervids, and pigs [32,33].Toxoplasmosis is an under-reported parasitic infection in Africa [34].Tere are limited studies on toxoplasmosis in SA; however, seropositive animals (cattle, sheep, and cats) have been reported [35].
Te development of persistent zoonotic transmission from initial spillover incidents requires the interaction of complex mechanisms [36].Conversely, there is general agreement that interaction between people and animals and their bodily fuids, whether directly or indirectly, is necessary for a successful interspecies transmission [36].In developing countries, 25% of the infectious disease burden is due to zoonotic diseases [37], as poverty raises the risk of zoonotic diseases spreading in communities where people are in close contact with livestock and wildlife [38,39].Te World Health Organization (WHO) reported an estimation of 600 million cases due to food-borne infections in 2010,while pathogenic bacteria were responsible for 350 million deaths of these cases [40].Livestock and wildlife play a major role in the economies of many developing countries by providing food, income, and employment for the communities [41].In low-income communities, livestock also serve as a source of wealth, means of transportation, and organic fertilizer for production [41].Terefore, the burden of infections in livestock may result in reduced productivity and reduction in the number of live animals due to increased death rates, abortions, and reduced meat and milk production [41].
Globally, there is a high demand on food due to the increasing human population [42].Te implication is that there will be high demand for animal protein.Tis means that more animals will be slaughtered to meet the demand.Abattoir surveillance of diseases in slaughtered animals using serological and cultural assays may be used to diagnose newly introduced diseases and monitor disease control and eradication eforts.Information generated from abattoir surveillance could provide an early warning system for impending epidemics or failures of intervention programs such as livestock vaccination against certain diseases, 2 International Journal of Microbiology thereby allowing early intervention eforts to prevent epidemic loss of animals and infections in humans.Te usefulness of data obtained from abattoirs during surveillance for selected diseases is however dependent on the accuracy of the data obtained [43,44].Tis study focused on investigating the presence of zoonotic diseases that are mainly asymptomatic except for causing abortion, namely, brucellosis, coxiellosis, and toxoplasmosis in slaughtered livestock at abattoirs in the Eastern Cape Province.Te objective was to determine the seropositivity of infectious zoonotic pathogens and the coexposure of the abovementioned diseases in slaughtered animals.), with a human population of more than six million [45].Tis area is surrounded by twelve registered abattoirs, including both high and low throughput abattoirs.

Selected Abattoirs.
In this study, seven abattoirs were randomly selected in the study area (Figure 1); however, only fve consented to participate.Tese comprised of high throughput (n � 3), slaughtering more than 20 livestock per species per day, and low throughput abattoirs (n � 2) slaughtering less than 20 livestock per species per day.Te abattoirs are located more than 100 km apart from each other, with the exception of one low throughput abattoir, being in close proximity to one of the high throughput facilities.Te managers of the selected abattoirs were notifed about the research project and their consent to participate in the study was obtained before the study commenced.

Sampling and Data Collection.
A randomly chosen subset of a single species was used to sample animals in a sequential manner.A total of 565 serum samples (280 cattle, 200 sheep, and 85 pigs) were randomly collected from fve abattoirs between 2020 and 2022.Terefore, a total of 160, 115, and 290 samples were collected in 2020, 2021, and 2022, respectively.In addition to serum sampling, tissues (liver, spleen, kidney, lymph nodes, and tonsils) were collected from corresponding carcasses.Tissue samples from seropositive animals were cultured for Brucella isolation.Isolated Brucella species were confrmed with PCR.Approximately 50 ml of unclotted blood was collected at the point of slaughter from the jugular vein into sterilized cups, with 5 ml of the blood aliquoted into yellow-capped vacutainer tubes.Information regarding the animals was obtained from the abattoir managers, which included the age, sex, breed, and the origin of the animals.However, in some abattoirs, the animal origins were not provided.Te blood samples were stored at 4-8 °C prior to serum separation.Te serum was aliquoted into 2 ml sample tubes, packaged in accordance with the National Road Trafc Act, 1996 (Act No. 93 of 1996), and transported to the Department of Veterinary Tropical Diseases, University of Pretoria, South Africa.

Sample Size and Data
Analysis.Te sample size was calculated using the following formula: where n is the sample size, P exp is the expected prevalence, L is the precision of the estimate (also called "the allowable error" or margin of error) which is equal to half the width of the confdence interval, Q = 1 − P exp , and Z is the (1 − α 2 ) percentile of a standard normal distribution [45]; for α = 0.05, Z = 1.96.Due to lack of recent data on seropositivity of Brucella spp., T. gondii, and C. burnetii in cattle, sheep, and pigs in Eastern Cape Province, 11% prevalence was used based on recent data from other provinces in South Africa to determine the sample size [11,35].Tis provided the sample size of 151 for each species which meets the required sample size per the calculation, except for the porcine samples as fewer pig animals were received by the abattoirs during the sampling period.Data were cleaned and managed in Microsoft Excel ® spreadsheet and the descriptive analysis (reporting the seropositivity, 95% confdence interval, and their associated P values) was performed using Stata V 14 (StataCorp, College Station, TX, USA).

Serological Testing.
Te criteria for Brucella seropositivity require the results of two or more of the techniques listed below for the valid confrmation of a sample.

Brucellosis Rose Bengal Test (RBT).
Te RBT was conducted using antigens and controls provided by Onderstepoort Biological Products (OBP).Te testing was conducted according to the World Organization for Animal Health (WOAH) protocol [46].Based on past validation tests, it has been determined that the RBT's diagnostic sensitivity and specifcity are 100.00%and 75.00%, respectively [47,48].Briefy, serum samples were removed from the freezer to the fridge to defrost overnight.Te samples were kept at room temperature prior to testing.A volume of 50 µl of each serum sample was added into each well, and the formation of bubbles was avoided.An equivalent volume of 50 µl of a positive control and a negative control were dispensed into separate wells as per the protocol.An equal volume of the anti-Brucella antigen was dispensed into each well containing the sera and positive and negative controls.Te plate was agitated on a rocker for 4 minutes.Samples were considered positive for RBT when a visible agglutination was observed.Te mean sensitivity and specifcity were 81.2% and 86.3% reported by Gall and Nielsen [49] when most tests were not credited for ISI 17025 and 9001, while Chisi et al. [50] reported diagnostic sensitivity and specifcity in SA bovine to be 95.8% and 100%.
International Journal of Microbiology

Brucellosis Complement Fixation Test (CFT). Te CFT was conducted at a South African National Accreditation System (SANAS) approved Serology laboratory at Agricultural
Research Council-Onderstepoort Veterinary Research (ARC-OVR) in South Africa according to the WOAH protocol.Te cut-of value for this test was ≥30 IU/ml as an indicator of infection.Te mean sensitivity and specifcity were 89.0% and 83.5% reported in [49], when most tests were not credited for ISI 17025 and 9001, while Chisi et al. [50] reported diagnostic sensitivity and specifcity in SA bovine to be 93.9% and 100%.

Brucellosis and Coxiella burnetii Enzyme-Linked Immunosorbent Assay (ELISA).
Te serological tests to detect the presence of antibodies against Brucella spp.and C. burnetii were confrmed using indirect enzyme-linked immunosorbent assay (iELISA).Each sample was assessed in duplicate to reduce variance.Te Brucella spp.and C. burnetii iELISA were conducted using the ID Screen brucellosis serum indirect multispecies (France) and ID Screen Q-Fever indirect multispecies kits (France), respectively.Both assays were performed as per manufacturer's instructions and the optical densities were read using the BioTek ELISA reader.Briefy, the frozen serum samples were stored at 4 °C overnight to defrost and brought to room temperature prior to testing.Te samples were vortexed and 10 µl of the samples were transferred into the C. burnetiiantigen-coated plates and the purifed Brucella LPS (lipo-polyssacharide)-coated plates, respectively.A positive and negative control supplied with each kit was added into the plate wells, and the plate was incubated at 25 °C for 45 min.Te plate was then washed three times with 300 µl of the wash solution (1X) using Bio-Rad PW40 microplate washer.Te conjugate bufer (100 µl) was then transferred into the washed plate and incubated at 25 °C for 30 minutes.Te above wash technique was repeated and 100 µl of the substrate solution was added into each well.Te plates were covered with foil and incubated in the dark for 15 minutes.Following the incubation, 100 µl of the stop solution was transferred into each well and the plates were read at 450 nm.Te results were interpreted as specifed by manufacturer's instructions.Te mean brucellosis iELISA sensitivity and specifcity were reported by 96.0% and 93.8% by [49], when most tests were not credited for ISI 17025 and 9001, while Chisi et al. [50] reported diagnostic sensitivity and specifcity in SA bovine to be 95.8% and 92.5%.

Toxoplasma gondii Mast
® Toxoreagent Test.A com- mercial latex agglutination test (LAT) produced by the Eiken Chemical Company of Japan, supplied by Davies Diagnostics (Pty.) Ltd., was used for detection of IgM and IgG antibodies against T. gondii and the reactions were performed using the U-shaped bottom microplates.Te samples were processed at dilutions of 1 : 16 to 1 : 2048.An agglutination titer of 1 : 64 dilution was considered as a positive cut-of level for T. gondii antibodies [34,51].

Isolation of Brucella spp. from Seropositive Livestock.
Te seropositive samples were processed according to set laboratory protocols in a bio-safety laboratory level (BSL) 2 + .About 200 μl homogenates of the tissues from seropositive animals (17/565) (liver, kidney, spleen, and lymph nodes) were inoculated onto the modifed CITA media [52] and incubated at 37 °C with 5.0% CO 2 for 5-14 days.Culture plates were considered negative and discarded following 14 days of incubation with no growth observed.Te mean sensitivity and specifcity were reported as 46.1% and 100%, respectively [49].Multiplex AMOS-PCR which identifed and diferentiated B. abortus (F-GAC GAA CGG AAT TTT TCC AAT CCC), B. melitensis (F-AAA TCG CGT CCT TGC TGG TCT GA), B. ovis (F-CGG GTT CTG GCA CCA TCG TCG GG), B. suis (F-GCG CGG TTT TCT GAA GGT GGT TCA), and IS711 (R-TGC CGA TCA CTT AAG GGC CTT CAT) was conducted as described [53].Te four species-specifc forward primers were used at a fnal concentration of 0.1 μM with 0.2 μM reverse primer IS711.PCR cycling condition consisted of an initial denaturation at 95 °C for 5 minutes followed by 35 cycles of 95 °C for 1 minute, 55.5 °C for 2 minutes, 72 °C for 2 minutes, and a fnal extension step at 72 °C for 10 minutes.Multiplex Bruce-ladder PCR was conducted as described [53] to diferentiate virulent Brucella spp.from S19 vaccine strain.

Results
A total of 565 sera samples were analysed with 49.56% cattle, 35.40% sheep, and 15.09% pigs.Te location of some animals was unknown, but it was established that the animals were transported from other provinces except for Western Cape Province, which requires an animal movement permit.Of all the 565 samples collected, 276 (48.85%) were females while 289 (51.15%) were males (Table 1).Within the 280 cattle samples, 162 (57.86%) were females which included 3 heifers and 118 (42.14%) were males which included 15 males less than 2 years old (Table 2).

Brucella DNA, Species Identifed from Cultures, and
Positivity.No pure Brucella cultures were obtained as slow growing Brucella colonies were overgrown by faster growing contaminant and thus PCR assays were used to identify Brucella and the species on isolates (Figure 2).Most Brucellalike colonies were subcultured two to three times and each time the colonies were tested with Brucella-specifc ITS PCR and then followed by AMOS-PCR assay but remained impure.
Brucella-specifc DNA was detected in 11 (64.71%) of the 17 seropositive animals using the AMOS-PCR assay.Of the 10 RBT seropositive cattle, 7/10 Brucella-specifc DNA was detected and identifed as B. abortus (Figure 2).Of the 4 iELISA seropositive cattle, 4/4 Brucella-specifc DNA was detected and identifed as B. abortus.Tus, of the 12 RBT or iELISA seropositive cattle, 3.57% (10/280) were seropositive and PCR positive for brucellosis.Bruceladder PCR was used to diferentiate between the feld and vaccine strain, of which it was verifed that there was no S19 vaccine strain isolated.Of the 4 seropositive sheep (RBT), 2/4 animals were culture positive for Brucella.Brucella-specifc DNA was detected and identifed as B. melitensis and B. abortus mixed infection for 1 animal while a B. abortus isolate was obtained from the second sheep.Tus, 1% (2/200) of the sheep was serology and PCR positive for brucellosis.Tere was no Brucella isolation from the seropositive pig sample.Brucella positivity of samples from Eastern Cape abattoirs were 4.3% (12/ 280) cattle, 1.0% (2/200)sheep, and 0/85 pigs based on at least two tests namely RBT, iELISA, or AMOS-PCR.

Discussion
Tis study investigated the presence of zoonotic infections causing abortions in livestock which included Brucella, C. burnetii, and T. gondii exposure, in apparently healthy livestock (cattle, sheep, and pigs).Despite the serious public health threat and economic impact these diseases have on livestock in South Africa, there is no surveillance program for these pathogens except for the partial bovine brucellosis scheme that focusses on high-risk bovines.Tus, abattoirs present an ideal passive surveillance opportunity for zoonotic infections in livestock.However, in a previous study, the trace-back of animals to a farm was limited especially with animals from feedlots and communal areas [11].We also investigated the coexposure and discussed the signifcance of these pathogen diagnostics in the relevant livestock species and possible health risks to humans handling these animals, such as abattoir workers and animal handlers.Tis study demonstrated the presence of antibody/antigen coexposure against (i) Brucella, C. burnetii, and T. gondii in  International Journal of Microbiology cattle, (ii) Brucella and T. gondii in sheep and cattle, and (iii) no coexposure observed in pigs.Coexposure of Brucella, T. gondii, and C. burnetii in cattle has not been reported in the literature.Although more than one-third of the serum samples were T. gondii positive in cattle, this result should be considered with caution due to the frequent lack of evidence of viable T. gondii infections in seropositive cattle.Tere are very few reports on naturally exposed cattle with positive T. gondii bioassays indicating active infections [54,55].Another South African study has reported codetection of C. burnetii and T. gondii (15.2%) in cattle from a communal farming area at the wildlife-livestock-human interface [56].Despite toxoplasmosis not commonly leading to reproductive pathologies in cattle, the organism results in the formation of cyst in the tissues posing a risk to public health if the contaminated meat is consumed undercooked [57].Also, in a recent publication from India, a 2.21% coexposure of C. burnetii and T. gondii was documented in dairy cattle, resulting in reproductive disorders [58].Coexposures of Brucella and C. burnetii in cattle and sheep are signifcant as they result in reproductive losses, and both have public health issues for humans in contact with infected animals [59,60].
Tis study observed an overall Brucella positivity of 4.3% and 1.0% in cattle and sheep, respectively.A study conducted from 2007 to 2015 had increased seropositivity by 6.31% in cattle, followed by 2.09% in sheep and 0.63% in pigs [61].Our study has reported 17 Brucella positive animals using all three serological tests, of which 66.7% were females.Tis high seropositivity rate in females poses zoonotic risks knowing the transmission route of the infection from animals to humans includes ingestion of contaminated milk [62].Tis fnding agrees with other studies which found defnitive diagnosis difcult in males [63,64].Te reason is that females are kept in the breeding herd for a substantially longer period than males, increasing their risk of infection [65].Although we are not privy to the exact animal origin details, there are 23 possible herds (farms) sampled between 2020 and 2022 at the abattoirs according to slaughter records.Te cattle made up 14 potential herds, while the sheep and pigs potentially made up 5 and 4 farms, respectively.Brucellosis is a herd disease so it is safe to assume that animals from the same herd would test positive.Te low seropositivity in cattle could, therefore, be due to the sample distribution according to origin.Of the 14 potential cattle herds tested, the 17 positive animals were distributed within 6 herds.Te 6 herds were predominantly from 2 provinces.
Although our seropositivity was low, our study has shown similar technique results to a study conducted by Kolo et al. [11] in Gauteng Province (SA) where they reported 11.0% RBT, 5.5% iELISA, and 2.0% CFT in cattle.Our study reported increased Brucella seropositivity of RBT (3.57%), followed by iELISA (1.42%) and CFT (0.36%).Our study had higher seropositivity in detecting antibodies against Brucella with the use of RBTcompared to iELISA and CFT.Te RBT is considered an exceptionally sensitive method to detect Brucella-specifc IgG1, IgM, and potentially IgG2 antibodies in cattle serum [66].Te RBT is primarily used as a standard test due to the high sensitivity of the assay, which might result in false-positive results [67].Although a previous South African study demonstrated no statistically signifcant diference in these tests' ability to diagnose Brucella, the RBT and ELISA showed an increased sensitivity and specifcity when combined [68].Also, these tests are validated on symptomatic animals or animals with documented pathology.Te CFT is highly specifc but may result in false negatives due to its low sensitivity [68], especially in asymptomatic animals.Our study further shows that the Brucella seropositivity is signifcantly higher in younger animals as compared to older animals.One would assume this could be due to latent infections in heifers being born from infected cows or due to vaccination with S19 as the study has shown increased positivity in females.However, there were only 3 heifers (<2 years) in our study.Te remaining positives were from young males (<2 years), which precludes vaccination since only females are vaccinated, thus suggesting a higher exposure of the pathogen in young cattle.Previous studies have reported an increased seropositivity in older animals than in younger animals Segwagwe et al. [67,69].We also observed higher positive cases among Bonsmara as compared to the Holstein breeds in cattle.Dairy herds consisting of Holsteins are farmed intensively, where reproductive issues are readily diagnosed and well documented [70].Te AMOS-PCR identifed B. abortus in cattle and a mixed infection of B. abortus and B. melitensis in sheep (64.71%) of the seropositive samples.
In this study, C. burnetii seropositivity of 26.43%, 15.00%, and 2.35% was observed in cattle, sheep, and pigs, respectively.A similar study conducted in Gauteng Province (SA) from slaughtered livestock at red meat abattoirs had comparable results with the present study where they reported a high seroprevalence of C. burnetii in cattle 9.40%, followed by sheep 4.30% and the least in pigs 0.90% [71].Te seropositive cattle were reported to have originated from eleven potential herds, while the sheep sampled potentially came from three and the pigs were potentially from two farms.Due to the infectious nature of C. burnetii, comingling of animals would lead to a higher seropositivity.Historically, there have been serological crossreactions for C. burnetii [72], which may result in false positives leading to an increased seropositivity.Tere are also cross reactions of C. burnetii with Coxiella-like endosymbionts (CLEs), making defnitive diagnosis diffcult [73]; thus, the results are similarly interpreted with caution.We detected antibodies against C. burnetii in 25.72% (71/276) females and 12.11% (35/289) in males.Previous research has demonstrated the presence of C. burnetii in semen [74], consequently suggesting that the higher seropositivity in females may be due to multiple females being involved with a single male during reproduction management [71].
Age was a statistically signifcant factor in our study considering that exposure to C. burnetii was higher among animals >2-3 years (38.60%) and >3 years (17.10%).Tese fndings may be due to increased possibilities of C. burnetii exposure with age.Te unique spore-like structures that International Journal of Microbiology C. burnetii produces are exceptionally resistant to environmental factors [75,76].Tese traits allow the bacterium to survive in the environment for up to a year, thus increasing the potential for exposure to livestock across seasons.In this study, cattle breed diferences were observed, with the highest seropositivity detected in cross-breed (35.71%), followed by Bonsmara (33.33%) and the least in Jersey (18.42%).Te Dorper breed (33.33%) had a higher seropositivity as compared to the Merino sheep breed (11.76%).
Te latex agglutination test (LAT) used for T. gondii is preferably used as a screening test.In comparison to the indirect immunological fuorescence test, the LAT assay has a comparable higher sensitivity [34].Te presence of IgM antibodies in serum are detectable about 1 week following infection and may persist for months or years [29].IgG antibodies indicate the existence of infection but give little information regarding when the infection frst occurred [29].We observed an increased T. gondii seropositivity in livestock between 2 and 3 years, 33.33% (19/57), followed by a decrease in animals older than 3 years, 19.05% (88/462).It should be noted that the sample numbers for livestock older than 3 years were larger than the preceding age group.Tis trend is contrary with studies reported in SA on sheep and West African cattle, sheep, and goat where the seroprevalence increased with the age of animals [34,77].Older animals, as opposed to young ones, are more likely to have a recurrence of T. gondii infection [82].According to other studies, older animals may be more susceptible to infection due to environmental exposure of infective oocysts that they ingest or inhale [83].By consuming uncooked meat infected with Toxoplasma cysts, scavenging cats around abattoirs can contract the infection and subsequently serve as sources of infection [83].Tis will increase the risk of infection among humans and livestock surrounding the abattoirs.

Conclusion
Tis study demonstrated the exposure to Brucella, C. burnetii, and T. gondii in apparently healthy cattle, sheep, and pigs where some animals showed coexposure to the pathogens.Te control of these selected zoonotic infections through vaccination programs is currently only possible for brucellosis; thus, the impact on public health is unknown.Furthermore, brucellosis detection is complicated by latency and limitation of serology tests and culture to detect chronic infected animals.Tests such as RBT are highly sensitive, but results can be confounded by the lower specifcity and false positives.While ELISA is highly specifc, the recommended cut ofs can lead to false negatives.Tese asymptomatic diseases pose a threat to humans who handle animals, such as abattoir workers, veterinarians, and animal technicians.Te detection of these pathogens largely depends on serological tests which are complicated by the risk of cross reaction to close-related species.Awareness regarding zoonotic infections is essential to sensitize laymen working in such industries to cultivate safe working practices and should be rolled out as part of control strategies.International Journal of Microbiology

Figure 1 :
Figure 1: Te Eastern Cape Province in South Africa is highlighted in purple in the South African map and selected abattoirs located within the circle in the Eastern Cape Province were sampled.

Figure 2 :
Figure 2: Te 2% agarose gel imaging demonstrating positive results by conventional PCR amplicons of 498 bp Brucella abortus target and 730 bp B. melitensis target.Lanes 1-7 are Brucella isolates from tissue samples, lane 8 (POS) is B. abortus, lane 9 (POS) is a mixed control of B. abortus and B. melitensis positive controls, and lane 10 is the negative control.

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[53]rnational Journal of Microbiology 2.7.Genomic DNA Extraction from Brucella Isolates.DNA was extracted from the suspected isolates for Brucella spp.screening.Tis was done using the Pure-Link Genomic DNA kit according to the instructions of the manufacturer.2.8.Brucella-Specifc PCR and Brucella Species Confrmation.DNA amplifcation for detection of Brucella region using genus-specifc 16S-23S rRNA interspacer region (ITS) was used for the detection of Brucella spp.[53].Briefy, a PCR master mix of 12 μl was prepared as follows: 6.5 μl DreamTaq polymerase, 0.3 μl (0.2 μM) forward primer, 0.3 μl reverse primer (0.2 μM), and 4.9 μl of nuclease-free water (Termo Fisher Scientifc, South Africa).From each sample, 3 μl of DNA was used in a 15 μl PCR reaction.Te mix was amplifed on a thermal cycler (Veriti 96 well) with a heated lid, preheated to 105 °C.Te PCR cycling condition consisted of 95 °C for 3 minutes, followed by 35 cycles of 95 °C for 1 minute, 60 °C for 2 minutes, 72 °C for 2 minutes, and a fnal extension of 72 °C for 5 minutes.Te target DNA region has a product size of 214 bp.

Table 2 :
Species results stratifed by gender and age.

Table 1 :
Serological results of Brucella spp. in livestock slaughtered from abattoirs in Eastern Cape Province.

Table 3 :
Serological evidence of Coxiella burnetii and Toxoplasma gondii in livestock slaughtered from abattoirs in Eastern Cape Province, SA.