Molecular prevalence, associated risk factors and phylogenetic evaluation of Theileria lestoquardi in the blood samples of small ruminants.

Raising small ruminants is the main source of income for farmers in Pakistan especially in rural areas of Dera Ghazi Khan in Punjab. Despite having large sheep population, the prevalence of intra-erythrocytic protozoa, Theileria (T.) lestoquardi, has never been reported from this area. This study was conducted to fill this knowledge gap and 333 blood samples of apparently healthy small ruminants (168 sheep and 165 goats) along with their epidemiological data were collected from Dera Ghazi Khan district during August till November 2022. The polymerase chain reaction (PCR) analysis amplified a 785 base pair amplicon specific for the Merozoite surface antigen (ms 1-2) gene of T. lestoquardi in 2 out of the 168 (3.3%) sheep blood samples, while no goat blood sample out of 165 was found to be infected with T. lestoquardi. DNA sequencing confirmed the presence of Theileria lestoquardi in both samples and phylogenetic analysis revealed that these amplicon resembled the partial ms 1-2 gene sequences detected in small ruminants from Pakistan, India Iran and Egypt. All the studied epidemiological factors (age, sex, breed, size of herd, dogs with herd, composition of herd, size of herd and Tick burden on sheep) were not found associated with the prevalence of T. lestoquardi. In conclusion, this study reports a low prevalence of T. lestoquardi infection in the Dera Ghazi Khan District of Punjab, Pakistan. The data generated from this work will help pave the way for the prophylactic detection and control of ovine and caprine theileriosis in the region.

Molecular prevalence, phylogeny and hematological impact of Toxoplasma gondii and Plasmodium spp. in common quails from Punjab, Pakistan.

This study investigates the molecular prevalence and phylogenetic characteristics of two prominent blood-borne pathogens, Toxoplasma gondii (T. gondii) and Plasmodium spp., in common quails (Coturnix coturnix) sampled from both wild (N = 236) and farmed (N = 197) populations across four districts (Layyah, Dera Ghazi Khan, Lahore, and Multan) in Punjab, Pakistan, during the hunting seasons from 2021 to 2023. Additionally, the impact of these pathogens on the complete blood count (CBC) of the hosts is examined. Out of 433 quails tested, 25 (5.8%) exhibited amplification of the internal transcribed spacer (ITS-1) gene for T. gondii, while 15 (3.5%) showed amplification of the Cytochrome b gene for Plasmodium spp. A risk factor analysis indicated that the prevalence of both pathogens was not confined to specific sampling sites or bird sexes (P > 0.05). District-wise analysis highlighted that hens were more susceptible to both T. gondii and Plasmodium spp. infections than cocks. Wild quails exhibited a higher susceptibility to T. gondii compared to farmed birds. Significant CBC variations were recorded in infected birds as compared to uninfected ones. BLAST analysis of generated sequences has confirmed the identity of recovered PCR amplicons as T. gondii and Plasmodium relictum. Phylogenetic analysis revealed that Pakistani isolates clustered with those reported from various countries globally. This study provides the first documentation of T. gondii and Plasmodium sp. infections in Pakistani quails, underscoring the need for detailed investigations across different regions to enhance our understanding of infection rates and the zoonotic potential of these parasites.

Molecular prevalence of vector borne bacterial pathogens in the blood samples of wild rodent species trapped from Saudi Arabia.

Order Rodentia is the most speciose among mammals and the members of this order are known to host more than 60 zoonotic diseases and rodents are a potential health threat to humans. This study was designed to report the molecular prevalence and phylogenetic evaluation of various blood borne bacterial pathogens (Anaplasma ovis, Anaplasma phagocytophilum, Anaplasma marginale and Bartonella spp.) in the blood samples of four wild rodent species [Meriones rex (N = 27), Acomys dimidiatus (N = 18), Myomys yemeni (N = 6) and Rattus rattus (N = 3)] that were trapped during August till October 2020 from Al Makhwah governorate in Saudi Arabia. Results revealed by 9/54 (16.6%) rodents amplified Msp4 gene and 2/54 (3.7%) rodents amplified rpoB gene of Anaplasma ovis and Bartonella spp. respectively. Anaplasma phagocytophilum and Anaplasma marginale were not detected among enrolled rodent species. Meriones rex was the most highly infected rodent species. DNA sequencing and BLAST analysis confirmed the presence of Anaplasma ovis and the Bartonella koehlerae in rodent blood samples. Phylogenetic analysis of both pathogens showed that Saudi isolates were clustered together and were closely related to isolates that were reported from worldwide countries. Risk factor analysis revealed that prevalence of both bacterial pathogens was not restricted to a particular rodent species or a rodent sex (P > 0.05). In conclusion, we are reporting for the very first time that Saudi rodents are infected with Anaplasma ovis and rodents can be infected with Bartonella koehlerae. Similar studies at large scale are recommended in all those areas of Saudi Arabia that are unexplored for the incidence and prevalence of bacterial pathogens among the rodents that are living near human dwellings in order to prevent bacterial infections in local people as well as in livestock.

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.

Simultaneous molecular detection of Anaplasma marginale and Theileria annulata in cattle blood samples collected from Pakistan-Afghanistan boarder region.

Theileria annulata (T. annulata) and Anaplasma marginale (A. marginale) are among the most extensively reported tick borne pathogens and are associated with huge economic losses worldwide. A total of 298 cattle blood samples were screened to report the presence of these two pathogens. The samples were collected from apparently healthy cattle (Achai, n = 155, Jersy, n = 88 and crossbred, n = 55) in Bajaur district of Khyber Pakhtunkhwa (KPK) during June and July of 2022. A total of 31 out of 298 cattle (10.4%) were found infected with T. annulata as PCR amplified a 156 base pair fragment from Tams-1 gene of T. annulata from their blood. While 16/298 animals (5.4%) were found infected with A. marginale as they amplified a 382 base pair fragment specific for msp5 gene of this bacterium. Three animals (1%) were found co infected. Cattle susceptibility to T. annulata infection was significantly higher than A. marginale infection (P < 0.001). Phylogenetic analysis revealed that Pakistani isolates of both detected pathogen clustered together and were closely related isolates from worldwide countries. Prevalence of T. annulata varied significantly among the sampling sites (P = 0.05) while no such association was observed for A. marginale among the tested cattle. Epidemiological data analysis revealed that none of the studied risk factors was found associated either with the prevalence of T. annulata or A. marginale (P > 0.05) among enrolled cattle. In conclusion, our study has revealed a relatively higher prevalence of T. annulata than A. marginale in cattle from the Bajaur district in KPK. This information is important for improving the productivity of the livestock sector, which is one of the main sources of income in the country. It is recommended that this data be taken into account for the development and implementation of effective tick control programs, as well as for the improvement of livestock management practices to prevent and manage TBDs in Pakistan.

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.

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.

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.

Quantitative analysis of lead in cows and buffaloes for health assessment.

The present study aimed to investigate the concentration of highly toxic heavy metal, lead (Pb), in cows and buffaloes of Sargodha district, Punjab, Pakistan. Samples of three major organs (spleen, lungs, bones) from five localities were obtained from two age groups (< 2 years, > 2 years) of each animal. With the confidence level of 95%, the investigated results demonstrated that lead concentration ranged from 0.28 to 1.12 mg kg-1 and 0.31 to 0.83 mg kg-1 in cows and buffaloes respectively. Out of the three tested organs, spleen contained the highest level of Pb (0.64-0.83 mg/kg) followed by lungs (0.28-1.12 mg/kg) and bones (0.36-0.87 mg/kg). Furthermore, a significant increase of Pb was observed in older animals. It was demonstrated that both age groups had Pb accumulation higher than the standard limit of 0.1 mg kg-1. Serving as bioindicators, the presence of toxic levels of lead in the animals indicated environmental pollution and possible risks for public health. High lead concentration in dietary commodities from Sargodha district calls for the sound management of hazardous waste. High levels of this toxic metal also indicate the possible health risks to human population of the areas in Sargodha. It is highly needed to address this metal contamination and its probable entry route into the food chain of the domesticated animals. This must be further investigated to prevent animals from lead exposure.

Retrospective Study of Cystic Echinococcosis (CE) Based on Hospital Record from Five Major Metropolitan Cities of Pakistan.

BACKGROUND: Human cystic echinococcosis (CE) is an important zoonotic parasitic disease. It is one of the NTDs having strong roots in Pakistan. There are limited studies on the prevalence of CE in Pakistan. METHOD: A retrospective study was conducted to determine the geographical distribution of human CE in Punjab (Lahore, Multan, and Faisalabad) and Sindh (Karachi and Hyderabad) provinces of Pakistan. Clinical records of infected patients from nine major hospitals were retrieved from 2008 to 2018. RESULTS: A total of 188 surgically confirmed cases were included from selected hospitals during the study period, which showed the annual frequency of 18.8 cases/year. The number of reported cases was higher in Sindh province (67.55%) compared to Punjab (32.45%). Total number of CE cases/city was higher in Karachi (47.34%) compared to Hyderabad city (20.21%), while in Punjab province, this frequency was higher in Lahore (25%) followed by Multan (5.32%) and Faisalabad (2.13%). Out of total 188 patients, 73 (38.83%) were males and 115 (61.7%) were females. In surveyed hospitals, high burden of infection was reported in young adults (21-30 years; 29.79%) followed by 21.81% and 17.02% in 31-40 and 41-50 age groups, respectively. Data retrieved from the hospital record of diagnosed cases of CE showed liver (36.17%, n = 68) as the most affected organ followed by lungs (26.06%, n = 49). In this survey, some unusual sites of cyst formation were also observed. CONCLUSION: Current study is a step forward towards filling the gap of knowledge for prevalence and regional distribution of hydatidosis in Pakistan reporting the CE cases in humans for the first time in Multan and Faisalabad.