Seroprevalence of Toxoplasma gondii, Neospora caninum and Trichinella spp. in Pigs from Cairo, Egypt.

Pork production is a niche economy in Egypt, and pigs are typically raised as backyard animals with no sanitary control, potentially exposing them to various pathogens. Commercially available ELISAs were used to detect specific antibodies to the food-borne zoonotic parasites Toxoplasma gondii and Trichinella spp., as well as to Neospora caninum, in serum samples of pigs slaughtered at Egypt's only licensed pig abattoir, the El-Bassatin abattoir in Cairo. Among the tested sera (n = 332), seroreactivity for T. gondii was 45.8% (95% confidence interval: 40.4-51.3), N. caninum was 28.0% (95% CI: 23.3-33.2), and Trichinella spp. was 1.2% (95% CI: 0.4-3.3). Mixed infection was only detected for T. gondii and N. caninum (18.7%; 95% CI: 14.7-23.4). The seroprevalence of T. gondii was significantly higher (p = 0.0003) in animals collected from southern Cairo (15 May city slum) than in eastern Cairo (Ezbet El Nakhl slum). Seroprevalence for N. caninum was higher in western (Manshiyat Naser slum; p = 0.0003) and southern Cairo (15 May city slum; p = 0.0003) than in that of eastern Cairo (Ezbet El Nakhl slum; p = 0.0003). Moreover, female pigs exhibited a higher rate of N. caninum antibodies than male ones (p < 0.0001). This study provides the first seroprevalence data for N. caninum in pigs in Egypt, and updates the prevalence of the zoonotic parasites Trichinella spp. and T. gondii.

First seroprevalence and molecular identification report of Brucella canis among dogs in Greater Cairo region and Damietta Governorate of Egypt.

Background and Aim: Given the rise in stray and imported dogs in Egypt over the past 5 years, it is surprising that no report of Brucella canis infection in dogs or humans has been documented in Egypt's published papers. This study aimed to detect the presence of antibodies against the rough (B. canis) and smooth Brucellae among dogs in Egypt and to characterize the Brucella species circulating in dogs. Materials and Methods: Blood samples (n = 449) were collected from owned and stray dogs in the Greater Cairo region (n = 309) and Damietta governorate (n = 140). The apparent, true, and total seroprevalence of canine brucellosis caused by B. canis infection were calculated using the 2-mercaptoethanol tube agglutination test (2-ME TAT) and rapid slide agglutination test (RSAT). We used the rose Bengal test (RBT) and the buffered acidified plate antigen test (BAPAT) to check the serum samples from dogs for the presence of antibodies against smooth Brucellae. Three polymerase chain reaction (PCR) assays - Bruce-ladder PCR, B. canis species-specific PCR (BcSS-PCR), and Abortus Melitensis Ovis Suis (AMOS)-PCR - were used to determine the Brucella species in the buffy coats of the serologically positive dogs. Results: The overall apparent and true prevalence of B. canis infection in dogs were estimated to be 3.8% and 13.2%. The estimated true prevalence in stray dogs (15%) was higher than in owned dogs (12.5%). The BAPAT and the RBT using smooth antigens revealed that 11 (2.4%) and 9 (2%) were positive. Bruce-ladder PCR targeting eryC, ABC, and Polysaccharide deacetylase genes was able to identify B. canis in nine out of 17 buffy coat samples. AMOS-PCR identified the eight undetermined Brucella species by Bruce-ladder PCR as Brucella abortus (n = 4) and Brucella melitensis (n = 4). To exclude the presence of Brucella suis, a one-step species-specific BcSS-PCR was performed and specifically amplified all B. canis DNA (n = 9) the same as did the Bruce-ladder PCR. Conclusion: To the best of our knowledge, this is the first report of B. canis detection in dogs in Egypt. Molecular identification of B. abortus and B. melitensis in the Egyptian canines highlights the role of stray dogs in brucellosis remerging in Brucellosis-free dairy farms. Brucella canis infection can be diagnosed specifically with the one-step BcSS-PCR. The obtained results set-an-alarm to the veterinary authorities to launch plans to control this disease in dogs.

Whole-genome sequencing (WGS) analysis of Brucella suis biovar 2 isolated from domestic pigs in Egypt for epidemiological and genetic diversity tracing.

In the current study, 14 Brucella suis biovar 2 (B. suis bv 2) strains isolated from slaughter pigs in Cairo were sequenced using Illumina technology to investigate genetic diversity, antimicrobial resistance (AMR) genes, and virulence-associated determinants. These strains were the first B. suis bv 2 isolates from Egypt. To place them in a global context, 92 genomes of B. suis were retrieved from the NCBI database and used for comparison. The in-silico analysis of MLST showed that all isolates have ST16. No resistome but 43 virulomes have been found without differences in distribution. The cgMLST classified the Egyptian B. suis strains into a complex type (CT) encompassing four distinct cgMLST sequence types. The closest relatives were strain B. suis 94/11 of an unknown origin and a Danish strain. Whole-genome sequencing analysis proved low diversity of Egyptian B. suis isolates; thus, a single introduction event is assumed. Investigation of a large number of B. suis isolates from different governorates is required to tailor control measures to avoid further spread.

Tracking the distribution, genetic diversity and lineage of Brucella melitensis recovered from humans and animals in Egypt based on core-genome SNP analysis and in silico MLVA-16.

Brucellosis is one of the most common neglected zoonotic diseases globally, with a public health significance and a high economic loss in the livestock industry caused by the bacteria of the genus Brucella. In this study, 136 Egyptian Brucella melitensis strains isolated from animals and humans between 2001 and 2020 were analysed by examining the whole-core-genome single-nucleotide polymorphism (cgSNP) in comparison to the in silico multilocus variable number of tandem repeat analysis (MLVA-16). Almost all Egyptian isolates were belonging to the West Mediterranean clade, except two isolates from buffalo and camel were belonging to the American and East Mediterranean clades, respectively. A significant correlation between the human case of brucellosis and the possible source of infection from animals was found. It seems that several outbreak strains already existing for many years have been spread over long distances and between many governorates. The cgSNP analysis, in combination with epidemiological metadata, allows a better differentiation than the MLVA-16 genotyping method and, hence, the source definition and tracking of outbreak strains. The MLVA based on the currently used 16 markers is not suitable for this task. Our results revealed 99 different cgSNP genotypes with many different outbreak strains, both older and widely distributed ones and rather newly introduced ones as well. This indicates several different incidents and sources of infections, probably by imported animals from other countries to Egypt. Comparing our panel of isolates to public databases by cgSNP analysis, the results revealed near relatives from Italy. Moreover, near relatives from the United States, France, Austria and India were found by in silico MLVA.

Isolation and molecular confirmation of Brucella suis biovar 2 from slaughtered pigs: an unanticipated biovar from domestic pigs in Egypt.

BACKGROUND: Brucella suis is a zoonotic pathogen with a serious impact on public health and the pig industry worldwide. Information regarding B. suis in pigs in Egypt is scarce. This study aimed to investigate the prevalence of B. suis in slaughtered domestic pigs at El-Basatin abattoir in Cairo, Egypt. A total of 1,116 domestic pigs slaughtered in 2020 were sampled for Brucella isolation and identification. Identified Brucella isolates were molecularly confirmed at species, and biovar levels using Bruce ladder PCR and Suis ladder multiplex PCR. Additionally, high-risk practices of 16 abattoir workers (4 veterinarians, 10 butchering and evisceration workers, and 2 scalding workers) were investigated using a pre-piloted structured questionnaire. RESULTS: Brucella isolates were recovered from 1.3% of examined pigs (n = 14) at consistently low rates (1.1-2.9%) across the year of sampling from February to December 2020. All isolates were confirmed as B. suis biovar (bv) 2. Remarkably, 92.9% (13/14) of isolates showed atypical ability to produce H2S and hence were considered as B. suis bv2 atypical phenotype. The prevalence was higher in males (1.8%) than in females (0.9). However, this difference was not significant (Odds ratio = 1.9; CI 95% 0.7 - 5.7; P = 0.2). No detectable pathological lesions were associated with B. suis bv2 infection in examined pigs. All strains were isolated from cervical lymph nodes, highlighting a potential oral transmission. High-risk practices were recorded among swine abattoir workers in this study: 75% do not wear gloves or disinfect their knives daily, and 18.8% were willing to work with open wound injuries. CONCLUSIONS: To the best of our knowledge, this is the first isolation of B. suis bv2 in Egypt. Detection of H2S producing B. suis bv2 atypical phenotype is alarming as it may result in misinterpretation of these isolates as highly human pathogenic B. suis bv1 in Egypt and possibly elsewhere. Further epidemiological tracing studies are crucial for the detection of the origin of this biovar. Including pigs in the national surveillance program of brucellosis, and an education program for swine abattoir workers about occupational risk of B. suis is a need in Egypt.

Tracking the Distribution of Brucella abortus in Egypt Based on Core Genome SNP Analysis and In Silico MLVA-16.

Brucellosis, caused by the bacteria of the genus Brucella, is one of the most neglected common zoonotic diseases globally with a public health significance and a high economic loss among the livestock industry worldwide. Since little is known about the distribution of B. abortus in Egypt, a total of 46 B. abortus isolates recovered between 2012-2020, plus one animal isolate from 2006, were analyzed by examining the whole core genome single nucleotide polymorphism (cgSNP) in comparison to the in silico multilocus variable number of tandem repeat analysis (MLVA). Both cgSNP analysis and MLVA revealed three clusters and one isolate only was distantly related to the others. One cluster identified a rather widely distributed outbreak strain which is repeatedly occurring for at least 16 years with marginal deviations in cgSNP analysis. The other cluster of isolates represents a rather newly introduced outbreak strain. A separate cluster comprised RB51 vaccine related strains, isolated from aborted material. The comparison with MLVA data sets from public databases reveals one near relative from Argentina to the oldest outbreak strain and a related strain from Spain to a newly introduced outbreak strain in Egypt. The distantly related isolate matches with a strain from Portugal in the MLVA profile. Based on cgSNP analysis the oldest outbreak strain clusters with strains from the UK. Compared to the in silico analysis of MLVA, cgSNP analysis using WGS data provides a much higher resolution of genotypes and, when correlated to the associated epidemiological metadata, cgSNP analysis allows the differentiation of outbreaks by defining different outbreak strains. In this respect, MLVA data are error-prone and can lead to incorrect interpretations of outbreak events.

Risk factors and Molecular genotyping of Brucella melitensis strains recovered from humans and their owned cattle in Upper Egypt.

Brucellosis is a zoonosis that has a devastating impact on the economy and public health, particularly in the Middle East, including Egypt. This study aimed to define risk factors associated with brucellosis in humans and in their cattle in Fayoum governorate - Upper Egypt. Also, molecular genotyping of recovered Brucella isolates from human cases and their cattle to assess the potential cross-species transmission in the study region. Data were obtained via double matched case-control studies for brucellosis in humans (106 cases and 160 controls) and in their cattle (78 cattle cases and 105 cattle controls). The results of multivariate regression analysis revealed that predictors of human brucellosis were animal-related occupations (OR 2.1, P 0.02), previous infection in other household members (OR 3.2, P 0.007), eating home-made soft cheese (OR 2.3, P 0.03), and exposure to cattle abortions (OR 6.9, P < 0.001). For cattle, predictors of brucellosis were maturity ≥2 years of age (OR 2.9, P 0.01), ≥2 animals reared by the same household (OR 3.7-6.9, P ≤ 0.001), and recent abortion (OR 15.2, P 0.01). Twelve Brucella isolates were recovered from eight human cases (7.5%, 8/106) and four cattle cases (6.2%, 4/65). All isolates were B. melitensis biovar 3. Analysis of the IS711 gene sequence revealed complete homology (100%) between isolates. Six virulence genes were utilized for virutyping: virB (100%), omp25 (100%), amiC (100%), ure (91.7%), wbkA (91.7%), and bvfA (75%). Virutyping revealed four virutypes: V1 (lack bvfA, 16.7%), V2 (harbored all genes, 66.7%), V3 (lack wbkA, 8.3%), and V4 (lack wbkA and ure, 8.3%). Repetitive extragenic palindromic PCR (REP-PCR) typing revealed two REP types. Combined REP-PCR/virulence genotyping revealed five different genotypes (G1-G5) for the detected isolates and a unique genotype for the reference strain (G6, B. melitensis bv3 Ether). Human and cattle isolates from the same household had matched genotypes. In conclusion, there were widespread risk factors among the cases studied. Health education for high-risk groups is essential for disease prevention, and combined REP-PCR/virulence genotyping is a quick tool for traceability, particularly in developing countries endemic with brucellosis as Egypt.

Molecular characterization and antimicrobial susceptibility testing of clinical and non-clinical Brucella melitensis and Brucella abortus isolates from Egypt.

Brucellosis is a highly contagious and incapacitating disease of humans, livestock and wildlife species globally. Treatment of brucellosis in animals is not recommended, and in humans, combinations of antibiotics recommended by the World Health Organization are used. However, sporadic antimicrobial-resistant (AMR) isolates and relapse cases have been reported from different endemic regions. In the current study, molecular characterization and antibiotic susceptibility testing using the microdilution method for 35 B. abortus and B. melitensis strains isolated from humans, milk and animal were carried out. Additionally, Next-Generation-Sequencing (NGS) technology was applied to confirm Brucella at the species level and investigate AMR and pathogenicity-associated determinants. MALDI-TOF seemed to be a rapid and reliable tool for routine identification of brucellae to the genus level; however, DNA-based identification is indispensable for accurate species identification. Brucella abortus strains were isolated from two human cases and a sheep. Such infections are uncommon in Egypt. Egyptian Brucella strains are still in-vitro susceptible to doxycycline, tetracyclines, gentamicin, ciprofloxacin, levofloxacin, chloramphenicol, streptomycin, trimethoprim/sulfamethoxazole and tigecycline. Probable (no CLSI/EUCAST breakpoints have been defined yet) in-vitro resistance to rifampicin and azithromycin was observed. WGS failed to determine classical AMR genes, and no difference in the distribution of virulence-associated genes in all isolates was found. Isolates of human and non-human origins were still susceptible to the majority of antibiotics used for treatment in humans. The absence of classical AMR genes in genomes of "resistant" Brucella strains may reflect a lack of information in databases, or resistance might not be encoded by single resistance genes. The One Health approach is necessary for tackling brucellosis. Continuous susceptibility testing, updating of breakpoints, assessing mutations that lead to resistance are needed.

Validation of real-time polymerase chain reaction versus conventional polymerase chain reaction for diagnosis of brucellosis in cattle sera.

BACKGROUND AND AIM: Different polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera. MATERIALS AND METHODS: One hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard. RESULTS: TaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%. CONCLUSION: A cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

MLVA fingerprinting of Brucella melitensis circulating among livestock and cases of sporadic human illness in Egypt.

Brucella melitensis is a serious public health threat, with human infection exhibiting acute febrile illness and chronic health problems. The present study investigated the genetic diversity and epidemiological links of the important zoonotic bacterium B. melitensis in Egypt using multilocus variable-number tandem repeat analysis (MLVA-16) including eight minisatellite (panel 1) and eight microsatellite (panel 2, subdivided into 2A and 2B) markers. A total of 118 isolates were identified as B. melitensis biovar 3 by classical biotyping and Bruce-ladder assay. Although B. melitensis is primarily associated with infection in sheep and goats, most of B. melitensis isolates in this study were obtained from secondary hosts (cattle, buffaloes, humans and a camel) suggesting cross-species adaptation of B. melitensis to large ruminants in Egypt. The MLVA-16 scheme competently discriminated 70 genotypes, with 51 genotypes represented by single isolates, and the remaining 19 genotypes were shared among 67 isolates, suggesting both sporadic and epidemiologically related characteristics of B. melitensis infection. Matching of local genotypes with representatives of global genotypes revealed that the majority of Egyptian isolates analysed had a West Mediterranean descendance. As this study represents the first comprehensive genotyping and genetic analysis of B. melitensis from different sources in Egypt, the information generated from this study will augment knowledge about the main epidemiological links associated with this bacterium and will allow a better understanding of the current epidemiological situation of brucellosis in Egypt. Ultimately, this will help to adopt effective brucellosis intervention strategies in Egypt and other developing nations.

Genetic variation of Brucella isolates at strain level in Egypt.

In this study, Multiple Locus Variable Number Tandem Repeat Analysis (MLVA-16) was performed on 18 Brucella isolates identified bacteriologically and molecularly (AMOS-PCR) as Brucella abortus (n = 6) and Brucella melitensis (n = 12). This was aimed to study the genetic association among some Egyptian Brucella genotypes isolated during the period from 2002 to 2013 along with the global genotypes database. MLVA-16 analysis for B. melitensis and B. abortus strains illustrates a total of 11, and 3 genotypes with 10 and 1 singleton genotypes, respectively. B. melitensis strains displayed greater markers diversity by VNTRs analysis of the 16 loci than B. abortus and this was attributed mainly to the diverging in panel 2B markers. B. melitensis genotype M4_Fayoum_Giza (3,5,3,13,1,1,3,3,8,21,8,7,5,9,5,3) was the only predominated genotype circulating between two different governorates. The most common B. abortus genotype, GT A3_Dakahlia (4,5,4,12,2,2,3,3,6,21,8,4,4,3,4,4), was present in three identical isolates. In phylogeny, Egyptian B. abortus bv1 genotypes were closely related to East Asian strain (for the first time), Western Mediterranean and Americas clonal lineages. B. melitensis local genotypes exhibit a genetic relatedness mostly to Western Mediterranean clonal lineage and one strain of Eastern Mediterranean clonal lineage. In conclusion, the geographic location is not the only factor stands behind the high genetic similarity of the Egyptian Brucella genotypes. These low variations may be a result of a stepwise mutational event of the most variable loci from a very limited number of ancestors especially during the transmission through non-preference hosts. The authors encourage the authorities in charge to establish pre-movement testing to reduce the risk of brucellosis spread.