A Pork Industry in the Backyard: An Analysis of Toxoplasma gondii Infection in Serbia's Pigs.

As pork is an important source for Toxoplasma gondii infection, we have analyzed T. gondii genotypes and toxoplasmosis prevalence in pigs in Serbia in the context of production statistics and economics to assess the specific risk to public health. Genotyping was performed using MnPCR-RFLP; T. gondii-specific IgG antibodies were detected using a modified agglutination test (MAT); and statistical data were extracted from official records and provided by government authorities. The results indicate that, from 2006 to 2021, the median number of annually slaughtered pigs was 5.6 million, yet only 36.1% were processed by abattoirs. The remainder were "backyard pigs" slaughtered on family farms and homesteads. Toxoplasmosis seroprevalence in market-weight (MW) pigs prior to 2006 was 15.2%, and was 15.1% in 2019. The seroprevalence in owned city cats, likely infected by livestock meat, was 33.2%. ToxoDB#1 was identified in pig tissues. The results indicate that backyard pigs are the backbone of the industry and provide as much as 60% of the pork in Serbia. The seroprevalence in pigs and city cats shows that farms are reservoirs for the parasite. Thus, innovative means of reducing T. gondii infection designed with backyard farmers in mind are needed to reduce the risk to public health.

Seroprevalence, Direct Detection and Risk Factors for Toxoplasma gondii Infection in Pigs in Serbia, and Influence of Biosecurity Measures.

Consumption of Toxoplasma gondii contaminated pork is a major risk factor for human infection. We thus conducted a cross-sectional survey on the seroprevalence of T. gondii infection in a representative sample of slaughter pigs from throughout Serbia and examined the influence of farm biosecurity-related risk factors on infection. In addition, direct detection of the parasite (by mouse bioassay) or its DNA was performed in the hearts of a subset of seropositive sows. The overall seroprevalence in the sample of 825 pigs as determined by the modified agglutination test (MAT) was 16.5%. Older age and inadequate rodent control were independent infection risk factors for pigs. In a subset of 581 pigs with complete biosecurity-related data, in addition to older age, smallholders' finishing type farms (as opposed to farrow-to-finish), multispecies farming, and origin from Western and Central and South-Eastern Serbia (vs. the Northern region), all increased the risk of infection, while the absence of disinfection boot-dips in front of each barn and Belgrade district origin (vs. the Northern region) were associated with a 62% and 75% lower risk of infection, respectively. Evidence of viable parasites was obtained in 13 (41.9%) of the 31 bioassayed sow hearts, of which by isolation of brain cysts in seven, by detection of T. gondii DNA in an additional four, and by serology in another two. Recovery of brain cysts mostly (5/7) from sows with a MAT titre of ≥1:100 indicates the risk for consumers. These results highlight the public health risk from pork consumption and point to mandatory use of professional rodent control services, abstaining from multispecies farming, keeping disinfection boot-dips clean and freshly refilled, as well as strict implementation of zoo-hygienic measures on smallholders' farms as specific farm biosecurity measures needed for its reduction.

Toxoplasma gondii Genotypes Circulating in Serbia-Insight into the Population Structure and Diversity of the Species in Southeastern Europe, a Region of Intercontinental Strain Exchange.

In Europe, Toxoplasma gondii lineage II is dominant, and ToxoDB#1 the most frequently occurring genotype. The abundance of lineage III genotypes varies geographically and lineage I are rare, yet present in several regions of the continent. Data on the T. gondii population structure in southeastern Europe (SEE) are scarce, yet necessary to appreciate the diversity of the species in Europe. To help fill this gap, we genotyped 67 strains from nine species of intermediate hosts in Serbia by MnPCR-RFLP, determined the population structure, and identified the genotypes using ToxoDB. A neighbor-joining tree was also constructed from the isolates genotyped on nine loci. While 42% of the total genotype population consisted of ToxoDB#1 and ToxoDB#2, variant genotypes of both lineages comprised 46% of the population in wildlife and 28% in domestic animals and humans. One genotype of Africa 4 lineage was detected in a human sample. Interestingly, the findings include one lineage III variant and one II/III recombinant isolate with intercontinental distribution, which appear to be moderately related to South American genotypes. Based on these findings, SEE is a region of underappreciated T. gondii genetic diversity and possible strain exchange between Europe and Africa.

In Vivo and In Vitro Virulence Analysis of Four Genetically Distinct Toxoplasma gondii Lineage III Isolates.

Toxoplasma gondii archetypes II and III are mildly virulent, yet virulence of variant strains is largely unknown. While lineage II dominates in humans in Europe, lineage III strains are present in various intermediate hosts. In Serbia, lineage III represents 24% of the population structure and occurs most frequently in domestic animals, implying a significant presence in the human food web. In this study, the virulence of four genetically distinct lineage III variants was assessed in vivo and in vitro. In vivo, two strains were shown to be intermediately virulent and two mildly virulent, with cumulative mortalities of 69.4%, 38.8%, 10.7%, and 6.8%, respectively. The strain with the highest mortality has previously been isolated in Europe and may be endemic; the strain with the lowest mortality matches ToxoDB#54, while the remaining two represent novel genotypes. Identical alleles were detected at ROP5, ROP16, ROP18, and GRA15. A set of in vitro analyses revealed proliferation and plaque formation as virulence factors. Higher levels of expression of ENO2 in intermediately virulent strains point to enhanced metabolism as the underlying mechanism. The results suggest that metabolic attenuation, and possibly stage conversion, may be delayed in virulent strains.

Computational image analysis reveals the structural complexity of Toxoplasma gondii tissue cysts.

Toxoplasma gondii is an obligate intracellular parasite infecting up to one third of the human population. The central event in the pathogenesis of toxoplasmosis is the conversion of tachyzoites into encysted bradyzoites. A novel approach to analyze the structure of in vivo-derived tissue cysts may be the increasingly used computational image analysis. The objective of this study was to quantify the geometrical complexity of T. gondii cysts by morphological, particle, and fractal analysis, as well as to determine if it is impacted by parasite strain, cyst age, and host type. A total of 31 images of T. gondii brain cysts of four type-2 strains (Me49, and local isolates BGD1, BGD14, and BGD26) was analyzed using ImageJ software. The parameters of interest included diameter, circularity, packing density (PD), fractal dimension (FD), and lacunarity. Although cyst diameter varied widely, its negative correlation with PD was observed. Circularity was remarkably close to 1, indicating a perfectly round shape of the cysts. PD and FD did not vary among cysts of different strains, age, and derived from mice of different genetic background. Conversely, lacunarity, which is a measure of heterogeneity, was significantly lower for BGD1 strain vs. all other strains, and higher for Me49 vs. BGD14 and BGD26, but did not differ among Me49 cysts of different age, or those derived from genetically different mice. The results indicate a highly uniform structure and occupancy of the different T. gondii tissue cysts. This study furthers the use of image analysis in describing the structural complexity of T. gondii cyst morphology, and presents the first application of fractal analysis for this purpose. The presented results show that use of a freely available software is a cost-effective approach to advance automated image scoring for T. gondii cysts.

Surface waters as a potential source of Giardia and Cryptosporidium in Serbia.

Giardiasis and cryptosporidiosis are recognized by the WHO as important emerging diseases of the 21st century. Symptoms are similar and include diarrhoea and vomiting, which may be severe, even life-threatening, for the immunocompromised and children under five years of age. Between 2013 and 2017, the Institute for Public Health in Serbia recorded 10 waterborne epidemics that manifested as gastrointestinal disease. Routine testing for enteropathogenic bacteria and viruses did not identify the aetiological agents of these outbreaks. As water is not examined for the presence of protozoa in Serbia, we performed a pilot study to analyse samples from four major rivers and their tributaries using a newly implemented methodology for detection of Giardia and Cryptosporidium, based on the ISO 15553:2006 standard. Using immunofluorescence microscopy, Giardia was detected in 10 out of the 31 samples, Cryptosporidium in five, while two samples were positive for both. Presence of G. duodenalis gDNA was confirmed by amplification of the ß-giardin gene in eight samples, of which one and two, respectively, were identified by RFLP as potentially zoonotic assemblages A and B. The results suggest that surface water in Serbia may be a potential source of infection and call for more in-depth studies using sophisticated molecular tools.

Detection and genotyping of Toxoplasma gondii in wild canids in Serbia.

To gain insight into the population structure of the protozoan parasite Toxoplasma gondii in wildlife in Serbia, multiplex nested PCR- RFLP was performed on T. gondii DNA positive samples of heart tissue of three wild canid species, including red foxes (Vulpes vulpes), golden jackals (Canis aureus) and grey wolves (Canis lupus). Out of 110 samples (28 red foxes, 72 golden jackals, 10 wolves), 30 (27.3%) were positive for T. gondii DNA, of which 24 were genotyped (nine red foxes, 13 golden jackals and two wolves). The results showed a large dominance of lineage II (n = 20, 83.4%) over lineage III (n = 4, 16.7%) strains. Lineage II comprised four archetypes, 14 variants and one which remained undetermined, while within lineage III there were three archetypes and one variant strain. However, a notable difference in diversity was observed among the examined canids, with eight (88.9%) of the nine strains detected in red foxes distinct, vs. six (46.1%) distinct strains of the 13 detected in golden jackals. Since anthropogenic food sources are a major reservoir of archetypal lineage II and III T. gondii strains, the results suggest that the golden jackal may be outcompeting the red fox, forcing it to hunt wild mammals and birds for sustenance, thereby increasing its chances of infection with variant strains. In addition to presenting the first data on the T. gondii population structure in wild canids in Serbia, this is the first report ever on T. gondii genotypes in golden jackals, a species with an increasing presence in Europe and another important reservoir of domestically circulating T. gondii strains.

Seroprevalence of Neospora caninum infection and associated risk factors in dairy cattle in Serbia.

Neosporosis in cattle is a globally important disease, causing abortions and significant economic losses if epidemic abortions occur. In Serbia, however, studies of Neospora caninum infection in cattle are few and are based on limited samples and/or from limited areas. We thus performed a nationwide study to examine the seroprevalence of N. caninum infection in a sample of cows randomly selected from dairy farms in 12 epidemiological units from throughout Serbia, as well as the possible transmission risk factors. Sera from a total of 1496 cattle were tested by competitive ELISA, and N. caninum-specific antibodies were shown in 7.2% animals (95% confidence interval CI, 6.6-7.9%), ranging from 2.2 to 12% across the epidemiological units. At least one seropositive animal was detected on 10.7% (95% CI, 9.7-11.8%) of farms, with a range of 5.9-25.9%. Logistic regression analysis showed that the single risk factor for infection in individual animals was keeping cows in loose-stalls (OR = 3.31, 95% CI = 1.95-5.60, P < 0.001). Risk factors for the presence of infection on farms also included housing in loose-stalls (OR = 18.49, 95% CI = 5.40-63.36, P < 0.001), and herd size > 100 animals (OR = 24.08, 95% CI = 3.85-150.50, P = 0.001). In view of the relatively low prevalence of infection showed at both the individual and farm level, this is the perfect time to undertake appropriate preventive measures to improve animal health and reduce economic losses.