Seroepidemiological and biomolecular survey on Toxoplasma gondii in Sardinian wild boar (Sus scrofa).

Toxoplasma gondii is a zoonotic parasite able of infecting all warm-blooded animals. Toxoplasmosis is one of the major foodborne diseases globally. The consumption of wild boar (Sus scrofa) meat from recreational hunting has been linked to outbreaks of human toxoplasmosis. The island of Sardinia (Italy) contains a large wild boar population, thus providing an opportunity to assess the distribution of Toxoplasma in this species and the associated risks of transmission to humans. A total of 562 wild boars were screened: heart and meat juice samples were tested for T. gondii DNA via nested-PCR and IgG anti-Toxoplasma by commercial ELISA. Anti-Toxoplasma IgG were detected in 24.6% (138/562) of animals, while 37.2% (209/562) of the heart samples were PCR positive. The prevalence of T. gondii antibodies and DNA highlights the potential role of wild boar as an important reservoir for this parasite. The study suggests that wild boar could play a significant role in spreading the parasite to humans. As wild boar numbers are increasing throughout their range, their potential role in transmitting toxoplasmosis should be communicated to stakeholders, and the impact of different population control methods on disease transmission should be thoroughly assessed to mitigate potential threats effectively.

Cobalt Uptake by Food Plants and Accumulation in Municipal Solid Waste Materials Compost-amended Soil: Public Health Implications.

One of the most pressing environmental issues is how to properly dispose of municipal solid waste (MSW), which represents both a substantial source of concern and a challenge. The current study evaluated cobalt (Co) accumulation in MSW, their uptake by different vegetables grown for two years, and related human health risks. Vegetables were grown in four different groups, such as one control (ground soil), and the remaining treatment groups (T1, T2, and T3) received varying concentrations of MSW. The analysis of Co was done through an atomic absorption spectrophotometer (AAS). Results revealed that the concentration of Co was higher in all the vegetables (n = 15) grown in soil supplemented with 75% MSW during 2nd growing year. Among all vegetables, the highest concentration of Co was observed in Solanum tuberosum at T3 during 2nd growing year. The pollution load index (PLI) value for vegetables during both growing years was more than 1 except in control soil. The findings indicated that the highest enrichment factor (EF) and hazard resilience index (HRI) value of 0.09 was present in S. tuberosum. Health index values for cobalt in the study were below 1. The HRI < 1 indicated that consumers do not face any immediate health risks. The investigation of Co concentrations in blood samples obtained from individuals residing in different areas contributes a human health perspective to the research. The findings indicate that the concentration of Co rises with an increasing proportion of MSW. While the metal levels in MSW-treated soil were not high enough to classify the soil as polluted, the results recommend that recycling MSW can substitute mineral fertilizers. Nevertheless, the presence of cobalt in MSW may directly affect soil fertility and could impact crop production and human health.

Molecular prevalence and phylogeny of Anaplasma marginale, Anaplasma ovis and Theileria ovis in goats and sheep enrolled from a hill station in Punjab, Pakistan.

Anaplasma marginale (A. marginale), Anaplasma ovis (A. ovis) and Theileria ovis (T. ovis) are among the most commonly reported intracellular tick borne pathogens that infect ruminants across the globe causing huge economic losses. This study aims to report the prevalence and phylogenetic evaluation of these three pathogens infecting sheep and goats (n = 333) that were enrolled from Fort Munro region in Pakistan by using msp1b, msp4 and 18S rRNA genes for A. marginale, A. ovis and T. ovis respectively. Results revealed almost similar infection rates in sheep and goats with an overall prevalence of 11% for A. marginale, 28% for A. ovis and 3% for T. ovis. Concurrent infection was also recorded, however, the number of animals infected with two pathogens (n = 24; 7.2%) was higher than infection with three pathogens (n = 2; 0.6%). Risk factor analysis revealed that sheep reared in small herds had higher A. marginale (P = 0.03) and A. ovis (P = 0.04) infection rates compared to those from large herds. In addition, it was observed that bucks (P ≤ 0.05) and tick-free goats (P ≤ 0.05) exhibited higher A. ovis infection rates than nannies. Phylogenetic analysis of all three pathogens showed that Pakistani isolates were clustered together and were closely related to previously deposited Pakistani isolates as well as with those that were reported from worldwide countries. In conclusion, we are reporting that Pakistani sheep and goats have A. marginale, A. ovis and T. ovis mediated infections and control measures should be taken against them to improve the productivity of the livestock sector.

Arsenic Levels and Seasonal Variation in Pasture Soil, Forage and Horse Blood Plasma in Central Punjab, Pakistan.

The present study aimed to determine the accumulation levels of arsenic in pasture soil, forage and animals. An atomic absorption spectrophotometer was used to determine the concentration of metals in the samples of soil, forage and blood plasma of horses. The level of arsenic ranged between 4.26 mg/kg (summer) and 4.66 mg/kg (winter) in soil samples and 2.67 mg/kg (summer) and 2.94 mg/kg (winter) in forage samples. In blood plasma samples, the mean arsenic (As) values varied between 1.38 and 1.52 mg/L. In the blood plasma samples, the mean As values varied between 1.38 and 1.52 mg/L. No statistically significant changes were observed for arsenic concentrations in plasma samples of horses for sampling seasons (p > 0.05). A positive correlation was observed for forage and blood plasma to a certain degree for arsenic but for other media, arsenic correlations were negative and insignificant. It is therefore suggested that regular monitoring of heavy metals in soils/plants/animals is essential to prevent excessive build-up of arsenic.

Influence of Industrial Wastewater Irrigation on Heavy Metal Content in Coriander (Coriandrum sativum L.): Ecological and Health Risk Assessment.

The primary objective of this study was to determine the heavy metal contents in the water-soil-coriander samples in an industrial wastewater irrigated area and to assess the health risks of these metals to consumers. Sampling was done from areas adjoining the Chistian sugar mill district Sargodha and two separate sites irrigated with groundwater (Site 1), and sugar mill effluents (Site 2) were checked for possible metal contamination. The water-soil-coriander continuum was tested for the presence of cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Ni), lead (Pb), and zinc (Zn). The mean concentrations of all metals were higher than the permissible limits for all studied metals except for Mn in the sugar mill wastewater, with Fe (8.861 mg/L) and Zn (9.761 mg/L) exhibiting the highest values. The mean levels of Fe (4.023 mg/kg), Cd (2.101 mg/kg), Cr (2.135 mg/kg), Cu (2.180 mg/kg), and Ni (1.523 mg/kg) were high in the soil at Site 2 in comparison to the groundwater irrigated site where Fe (3.232 mg/kg) and Cd (1.845 mg/kg) manifested high elemental levels. For coriander specimens, only Cd had a higher mean level in both the groundwater (1.245 mg/kg) and the sugar mill wastewater (1.245 mg/kg) irrigated sites. An estimation of the pollution indices yielded a high risk from Cd (health risk index (HRI): 173.2), Zn (HRI: 7.012), Mn (HRI: 6.276), Fe (HRI: 1.709), Cu (HRI: 1.282), and Ni (HRI: 1.009), as all values are above 1.0 indicating a hazard to human health from consuming coriander irrigated with wastewater. Regular monitoring of vegetables irrigated with wastewater is strongly advised to reduce health hazards to people.

Laboratory associated zoonotic parasitic infections: a review of main agents and biosecurity measures.

Laboratory workers are exposed to the risk of acquiring infections due to the manipulation of infectious materials. The biological hazard for researchers is seven times higher when compared with hospital and public health laboratory workers. Despite the implementation of standardized practices to control infections, multiple cases of Laboratory Associated Infections (LAIs) usually go unreported. There has been a lack of comprehensive epidemiological data regarding the situation of LAIs for parasitic zoonosis and besides, the available sources are not completely updated. Since most accounts of laboratory infections are organism-specific, this study has focused on common pathogenic/zoonotic species handled at parasitological laboratories and summarising the standard biosecurity protocols for the infectious agents. The main characteristics of Cryptosporidium spp., Entamoeba spp, Giardia duodenalis, Toxoplasma gondii, Leishmania spp., Echinococcus spp., Schistosoma spp., Toxocara canis, Ancylostoma caninum, Strongyloides stercoralis are considered in this review in order to assess the potential risk of developing occupational infections in the workplace along with stating prevention and prophylactic measures for each species. It was concluded that the LAIs from these agents can be prevented by using personal protective measures and good laboratory practices. However, further studies are necessary to better understand the environmental resistance of cysts, oocysts and eggs, with a view to select the most suitable disinfection methods. Furthermore, it is fundamental to constantly update epidemiological data of infection acquired by laboratory workers, to develop accurate risk indicators.

First report of Echinococcus ortleppi and genotype G6 of E. canadensis cluster from southern Punjab, Pakistan and a global overview on genetic structure and host adaptation of E. ortleppi.

Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato, is a neglected tropical disease known mainly for its zoonotic nature. CE is endemic to Pakistan, however, the disease is not given due consideration and millions of people remain at health risk. This study was undertaken to assess the species and genotypes of E. granulosus sensu lato in sheep, buffaloes and cattle, brought to slaughterhouses of two major cities (Multan and Bahawalpur) of south Punjab, Pakistan. A total of 26 hydatid cyst specimens were characterized through complete cox1 mitochondrial gene (1609 bp) sequencing. Species and genotypes of E. granulosus sensu lato discovered in the southern Punjab consisted of E. granulosus sensu stricto (n =21), E. ortleppi (n=4) and genotype G6 of the E. canadensis cluster (n=1). Of E. granulosus s.s. isolates, the genotype G3 was predominantly involved in causing infections to the livestock of this region. Since all of these species are zoonotic, wide and effective surveillance studies are required to ascertain the risks to human population in Pakistan. Additionally, a global overview on cox1 phylogenetic structure of E. ortleppi was carried out. Despite widespread occurrence, the species is mostly limited to the southern hemisphere. The highest burden has been reported in South America (62.15%) and Africa (28.44%) and by far the most common host is cattle, accounting for >90% of cases.

Taenia multiceps coenurosis: a review.

Taenia multiceps is a taeniid cestode that inhabits the small intestines of both wild and domestic carnivores. The larval stage, Coenurus cerebralis, is typically found in the central nervous system (CNS) of a wide range of livestock and, to a lesser extent, in the extra-cerebral tissues of sheep and goats. This review covers all aspects of the life cycle of T. multiceps and its epidemiology, molecular characterization, pathogenesis, diagnosis, therapy, control and zoonotic potential. Coenurosis caused by the larval stage of T. multiceps has a worldwide distribution and is often fatal in intermediate hosts, which can result in substantial economic losses in livestock farming. Molecular characterization using the mitochondrial genes cytochrome c oxidase subunit 1 and nicotinamide adenine dinucleotide dehydrogenase subunit 1 of different T. multiceps populations has revealed significant genetic variation and the presence of three major haplotypes. The disease mostly affects young sheep and is referred to as either acute or chronic coenurosis. Acute coenurosis occurs as a result of oncospheres migrating through the CNS, while chronic coenurosis occurs as a consequence of the coenurus maturing, which causes displacement and pressure atrophy of brain tissue. Non-cerebral coenurosis has been most commonly reported in goats. The best diagnostic method for cerebral coenurosis involves the interpretation of clinical signs with accurate localization of the cyst using diagnostic imaging techniques. A vaccine based on recombinant oncosphere antigens has proved to be an effective tool against T. multiceps infection in sheep. Additionally, use of anthelmintics during the parasite's migration stages reduces the development of cysts in the sheep brain. Surgery is considered the most effective method for the treatment of cerebral coenurosis in small ruminants, but is often not carried out because of the limited finances of many sheep and goat breeders. However, coenurosis can also be controlled effectively through preventative measures, such as anthelmintic treatment of dogs and the proper disposal of intermediate host carcasses. The parasite is also zoonotic, and cases of coenurosis have been reported in humans with coenuri located in the brain, spinal cord and eyes.

Genetic structure and phylogeography of Echinococcus granulosus sensu stricto genotypes G1 and G3 in Pakistan and other regions of the world based on nad5 gene.

Pakistan is a neglected endemic focus for Echinococcus granulosus sensu lato, a zoonotic parasite species complex with the ability to infect wide spectrum of hosts. Wide gaps exist in literature for etiological agents of cystic echinococcosis (CE) in Pakistan due to a very low number of studies on identifying the exact genotypes involved in epidemiological manifestation of this disease. Focusing on transmission patterns and epidemiological dynamics, this study aimed at investigating infective genotypes among the cattle population of south Punjab, Pakistan, employing a mitochondrial marker nad5 (680 bp). Nucleotide sequences retrieved from 28 hydatid cyst isolates displayed considerable intraspecific variation revealing the existence of G3 and G1 strains of Echinococcus granulosus sensu stricto. The G3 genotype emerged as the predominant cause (78.57%) of hydatidosis in cattle. Apart from this, to understand phylogeographical relations, homologous nucleotide sequences of the partial nad5 gene from six major regions of the world were employed in the population genetics analysis to have an insight into genetic variability and demographics of G3 genotype in particular. Diversification of G3 and its haplotypes in Pakistan (n = 11) and other regions of the world (India, Iran, Turkey, Italy and France) was demonstrated. It was further demonstrated that the South Asian population (Pakistan and India) was highly differentiated from the other regions. It could, therefore, be speculated that G3 is diverging and expanding its population with South Asia as the main focal point.

Human Cystic Echinococcosis in Lebanon: A Retrospective Study and Molecular Epidemiology.

PURPOSE: Human cystic echinococcosis (CE) is a zoonotic parasitic disease that constitutes a public health challenge and a socio-economic burden in endemic areas worldwide. No specific surveillance system of CE infections in humans exists in Lebanon. The incidence and trends over time have not been documented. The current study aimed to assess the demographic and epidemiologic features of human CE surgical cases over a 14-year period in the five main regions of Lebanon. METHODS: From 2005 to 2018, a total of 894 surgically confirmed cases of hydatidosis were recorded from five anatomy and pathology laboratories. RESULTS: The mean annual surgical incidence was 1.23/100,000 inhabitants. Over the span of these years, the incidence increased from 0.53 to 1.94 cases/100,000 inhabitants in 2005 and 2018, respectively. CE is present in Lebanon with an uneven distribution from one region to the other with higher prevalence in Bekaa (29.0%), a rural area where sheep raising is widespread. Human CE cases were more common in females (60.1%) than in males (39.9%) and a high burden of infection was reported for the age group of 30-39 years. Besides, 66.7% of the cases expressed only liver complications whereas, 20.5% showed predilection towards lungs. The 7.8% of cases presented cysts in other organs, and 1.3% showed multiple localizations. Additionally, predominant involvement of Echinococcus granulosus sensu stricto was recorded in human infections. Comparison of Echinococcus granulosus s.s. populations from different Mediterranean countries also revealed high gene flow among this region and sharing of alleles. CONCLUSION: The current study is a step forward to fill the gap of knowledge for the hydatidosis in Lebanon where the lack of epidemiological data and control measures have resulted in higher incidence of human CE.

First molecular description of Macracanthorhynchus hirudinaceus in wild boars from Italy with pathomorphological and epidemiological insights.

Macracanthorhynchus hirudinaceus is a zoonotic parasite affecting suids worldwide which are the definitive hosts for this helminth species. Macracanthorhynchus hirudinaceus is of significant economic and management concern due to its pathogenicity, causing intestinal obstruction and perforation in the definitive hosts. Current study is the preliminary investigation from Sardinia, Italy, reporting the pathomorphological findings and molecular characterization of M. hirudinaceus in the wild boars (Sus scrofa meridionalis). A total of 59 wild boars were examined showing acanthocephalan infection in 8 (13.6%) animals. In total, 49 parasites were collected with a mean intensity of 6.1. Comparatively higher infection levels were observed for males (16.7%) and young boars (14.3%); however, these epidemiological differences were statistically non-significant. Histopathological examination revealed the presence of a variable number of nodules (∼5 mm) in the intestine of M. hirudinaceus infested animals surrounded by a hyperemic-hemorrhagic halo. Several parasites were recovered from the intestinal lumen attached by the means of characteristic hooks showing necrosis in muscle layers. A moderate number of plump reactive fibroblasts and lesser numbers of fibrocytes were embedded with and at the borders of the inflammatory nodules in a moderate amount of homogeneous intensely eosinophilic fibrillary material rupturing the cell membrane. For molecular characterization, six isolated worms were amplified for the partial mitochondrial cox1 gene showing distinct interindividual variations. This first pathological and molecular description from southern Europe provided new knowledge about the diffusion of M. hirudinaceus in wild boars, furthering the research into the origin and transmission status of M. hirudinaceus in endemic localities.

Practical guide for microscopic identification of infectious gastrointestinal nematode larvae in sheep from Sardinia, Italy, backed by molecular analysis.

BACKGROUND: Gastrointestinal nematodes (GIN) are ubiquitous in small ruminant farming, representing a major health and production concern. Given their differences in pathogenicity and the current problems regarding anthelmintic resistance, specific diagnosis of GIN is of significant importance. At present, the most widely applied method for this entails culture and microscopic analysis of third-stage larvae, allowing for identification at least to the genus level. Overall, a variety of keys for microscopic analysis have been published, showing substantial variation. Given this fact, this study aimed to produce a practical and updated guide for the identification of infective ovine GIN larvae. METHODS: Using existing keys and protocols, a total of 173larvae of the most common species/genera of ovine GIN from pooled faecal samples from Sardinia (Italy) were identified and extracted, and further individual molecular identification was performed. Morphometric and morphological data as well as high-quality photographs were collected and combined to produce the final guide. RESULTS: GIN microscopically and molecularly identified during this research include Trichostrongylus spp., Teladorsagia circumcincta, Haemonchus contortus, Cooperia curticei, and Chabertia ovina. Based on microscopic analysis, 73.5% of the larvae were correctly identified. Based on sheathed tail length, 91.8% were correctly classified into their respective preliminary groups. CONCLUSIONS: It is crucial for the microscopic identification of infectious GIN larvae to examine each larva in its entirety and thus to take multiple characteristics into account to obtain an accurate diagnosis. However, a preliminary classification based on sheathed tail length (resulting in three groups: A, short; B, medium; C, long) was found to be effective. Further identification within group A can be achieved based on the presence of a cranial inflexion, caudal tubercles and full body measurements (Trichostrongylus spp. < 720 µm, T. circumcincta ≥ 720 µm). Larvae within group B can be differentiated based on sheathed tail morphometry (H. contortus > 65 µm, C. curticei ≤ 65 µm), the presence of cranial refractile bodies, total body length measurements (H. contortus ≤ 790 µm, C. curticei > 790 µm) and shape of the cranial extremity. Finally, all characteristics proposed for the differentiation between Oesophagostomum spp. and C. ovina larvae (group C) were found to have considerable restrictions.

Genetic diversity and transmission patterns of Echinococcus granulosus sensu stricto among domestic ungulates of Sardinia, Italy.

Cystic echinococcosis (CE), a parasitic zoonosis of public health and economic concern, is highly endemic in Sardinia, Italy. The study involved examining the intraspecific variability and demographic structure of Echinococcus granulosus sensu stricto (s.s.) in common hosts of this parasite. Molecular surveillance included the fragment amplification of a partial mitochondrial gene, cox1 (750 bp), for a total of 69 isolates derived from sheep (n = 52), cattle (n = 11), pigs (n = 4), and goats (n = 2). It was ascertained that E. granulosus s.s. was the primary agent of infection among these ungulates and G1 genotype was highly prevalent (79.71%). Considerable intraspecific variation was found, revealing the existence of 22 haplotypes with relatively high haplotype (0.8555 ± 0.033) and low nucleotide diversities (0.00281 ± 0.00030). Population demographics indicated an expanding parasitic population signifying negative deviation from neutrality indices. Little genetic differentiation was found between the subpopulations of E. granulosus s.s. in the island. Moreover, the geographic dispersal of genotypes G1 and G3 also indicated similarity between Sardinian and mainland Echinococcus granulosus s.s. populations reaffirming the sympatric occurrence and efficient transmission of G1 and G3 genotypes. Molecular survey of CE has the potential to yield baseline information on the infective genotypes among the intermediate hosts and helps in devising suitable control strategies for curtailing the disease.

Cystic echinococcosis in sheep and goats of Lebanon.

Cystic echinococcosis (CE), a zoonotic disease caused by the species complex of Echinococcus granulosus sensu lato (s.l.), is endemic in Middle East and the Mediterranean basin, where pastoral activity is widespread. Despite the chronic endemicity of the disease in Lebanon and neighbouring countries, recent data are scant. The objectives of this survey were to evaluate the current epidemiology of CE in Lebanon, investigate the prevalence and determine the infecting genotypes in locally raised sheep and goats. A multidimensional approach combining post-mortem inspection of slaughtered animals and molecular diagnosis of the parasite was conducted to this end. From 2018 to 2020, 62.9% of the sheep and 20.9% of the goats were found positive for CE. The presence of hydatids varied between organs, showing higher prevalence in the liver of sheep vs the lungs of goats, however, a higher fertility rate of hydatid cyst was observed in lungs for both animals. Molecular diagnosis based on partial mitochondrial cox1 gene (795 bp) showed that the majority of isolates were identified as E. granulosus sensu stricto (98.7%) and only one isolate from goat was identified as Echinococcus canadensis (genotype G7; 1.3%). Echinococcus granulosus s.s. population among the sheep and goats was represented by 22 haplotypes having very little genetic differentiation and relatively moderate haplotype diversity. Population demographics explored through neutrality indices suggested expanding population within the intermediate hosts. These results document the high prevalence of CE in the livestock of Lebanon and reveal for the first time the presence of three different genotypes G1, G3 and G7.

Mineral availability in soil and fodders affecting blood profiles in Nili-Ravi dairy buffaloes.

A mineral assessment study for dairy buffaloes, Nili-Ravi breed, was carried out seasonally at silvopasture farm at Sargodha, Pakistan. Sampling for soil, forage, and buffalo serum was done seasonally for a period of 1 year for mineral (P, Mg, Na, K, Ca) evaluation. In the study, experiments on forages showed significant differences in mean concentrations of Mg and P with changing seasons, whereas the values for Ca, Mg, Na, and K did not vary significantly. Three groups of buffaloes were checked for mineral concentration in blood serum, viz., calves, lactating and non-lactating. Ca mean concentration varied significantly for non-lactating buffaloes and calves between two seasons. Na mean level showed a significant difference for two sampling seasons among calves and lactating buffaloes. Mean level for Mg only differed significantly in serum of non-lactating buffaloes between the two seasons. Mean levels for Ca and P in soil and forage remained higher than the optimal level, whereas soil Na was below the critical level. K concentrations in soil and blood serum were above the critical level, while Mg levels remained below the optimal range in soil, forage, and blood serum. K concentration in forage remained below the optimal level. All buffalo categories displayed lower levels of Ca and P in serum. Na concentration in forage and serum exceeded the critical level. Furthermore, Ca yielded significant and positive correlation between soil and serum level, whereas K had significant but negative correlation for soil-forage and forage-serum. Overall, it was determined that P deficiency existed among buffaloes, which could be improved via supplementation. Similarly, K deficiency and Mg deficiency were exhibited for forage and soil respectively which could be countered through addition of fertilizers rich for these minerals. In brief, the mineral utilization of buffaloes is affected by reduced availability of essential nutrients and may result in lower productivity. It is suggested to add mineral supplementation in addition to natural diet of buffaloes to enhance the productivity of these animals.

Comparative study of forage toxic metals of conventional versus non-conventional pastures in relation to animal mineral nutrient allowance.

At present, the forage production is the foundation of beef cattle and sheep industries in graziery. This study was designed to assess the toxic metal composition of the conventional pastures and non-conventional pastures in consideration of mineral needs of ruminants. At all the surveyed sites, these mean soil metal concentrations were measured to be higher or lower than the referred threshold levels. Specifically, these concentrations of Co, Pb, and Cr were observed as much higher than the referred threshold levels of 0.01-0.06 mg/kg (Co), 0.05 mg/kg (Pb), and 0.02 mg/kg (Cr). On the contrary, the mean soil concentrations of Cd were observed to be relatively lower than the threshold levels of 0.50-10 mg/kg at all sites. The estimated mean concentrations of Co, Cd, Pb, and Cr in both conventional and non-conventional forage species were measured to be lower than the referred threshold levels. The supplementation of these metals for livestock grazing from pasture was unnecessary based on these findings. However, further research should be conducted for ecological safety and conservation of ruminant forages, so that both the conventional and non-conventional pastures are employed as livestock friendly consuming forages.

Comprehensive Account on Prevalence and Characteristics of Hydatid Cysts in Livestock from Pakistan.

Pakistan is at intersection of hyperendemic regions for hydatidosis. Current study aimed to determine the prevalence of hydatid cysts and cyst characteristics in different intermediate hosts (sheep, goats, cattle and buffaloes) across the 4 provinces of Pakistan. A total of 991 sheep, 1,478 goats, 1,602 cattle and 1,343 buffaloes were examined for presence of hydatid cysts during 2 years (January 2016-December 2018). Differences in frequency of hydatidosis were observed with highest overall prevalence in buffaloes (11.9%) and sheep (11.5%). Highest prevalence and burden of infection were observed in older age animals (23.8%, 9.78±0.49) and females (26.5%, 12.53±0.67). Data for seasonal prevalence alluded to year-round presence of disease with non-significant statistical difference. Organ predilection indicated liver as the most preferred site of cyst localization followed mainly by lungs. An over-dispersion pattern was observed in all infected animals as majority of cysts belonged <10 cysts per infected host category. Highest percentage of fertile cysts was observed in liver of sheep. Interestingly, solitary form of cysts had higher fertility rate than multiple form. Amid lack of data and wide gap of knowledge, this study would try to fill up the lacunae regarding this neglected tropical disease. Extensive rearing of livestock, unregulated official slaughter and home slaughtering have played role in adaptability of E. granulosus in Pakistan.

Genetic variability and diversity of Echinococcus granulosus sensu lato in human isolates of Pakistan based on cox1 mt-DNA sequences (366bp).

Cystic echinococcosis (CE) is a cyclo-zoonotic disease endemic to Pakistan, however, phylogeography and etiology of Echinococcus granulosus from human host has remained unexplored so far. The current study was carried out to estimate population structure and appraise the genetic variability of E. granulosus in two major provinces of Pakistan, Sindh and Punjab. 94 isolates were subjected to sequence analysis from histopathologically confirmed CE patients using a fragment of mitochondrial cytochrome c oxidase subunit 1 gene (cox1; 366bp). Three genotypes of E. granulosus sensu lato, G1 (57.44%), G3 (41.48%) and G6 (1.06%), were inferred to exist in human patients. Molecular diversity indices for E. granulosus sensu stricto manifested the presence of 14 haplotypes among the regional populations forming distinct clades with sheep (G1) and buffalo (G3) strains. The haplotype network displayed a double clustered star like feature with PK-H1c (37.63%) and PK-H9c (32.25%) as most dominant haplotypes displaying low genetic differentiation between the two geographic regions. Neutrality indices (Tajima's D and Fu's Fs) were negative for all populations indicating population expansion across Pakistan. The sympatric presence of G1 and G3 genotypes among humans in endemic focus of Pakistan confirms the zoonotic potential of E. granulosus s.s. with special emphasis on the G3 strain which emerged as a dominant cause of CE in humans compared to other global studies, where only G1 is linked to the majority of CE cases. Data from this study will prove to be a keystone for devising robust regional control strategies for human hydatidosis and investigating potential pathogenicity of E. granulosus s.l. haplotypes and their clinical manifestations in Pakistan.

Comprehensive study based on mtDNA signature (nad1) providing insights on Echinococcus granulosus s.s. genotypes from Pakistan and potential role of buffalo-dog cycle.

Pakistan has long been considered neglected endemic region for Echinococcus granulosus. Limited surveillance studies have failed to epidemiologically draw complete picture on geographical presence and etiological agents of cystic echinococcosis. Amidst such lacunae, current study explored main transmission routes of this disease through molecular characterization of hydatid cyst isolates obtained from sheep (n = 35), goats (n = 26), cattle (n = 30) and buffalo (n = 30) from the four provinces of Pakistan. Two strains of E. granulosus sensu stricto, G1/G3, and their haplotypes were observed to be cycling in sympatry in the domestic ungulate populations. G3 genotype had higher prevalence (66.94%) in the hosts compared to G1 genotype (33.06%) which was not surprising, considering the large buffalo population in Pakistan. Haplotypic analysis revealed presence of 9 different haplotypes configured in a double clustered network with two centrally positioned haplotypes referred to as G3 (PKH1) and G1 (PKH6). Population demographics and genetic variability indices suggested expanding parasitic population in multiple host spectrum. Elucidating local transmission patterns of E. granulosus sensu stricto, buffalo-dog cycle emerged as one of the dominant causes of G3 dispersal in contrary to other global studies. Adaptability of G3 to environmental conditions of Pakistan and high affinity for buffaloes emphasize on heterogeneous nature of this strain in contrast to G1. However, more studies involving larger datasets and mitochondrial sequences could confirm this hypothetically formulated inference.