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Livestock industry is an essential part of Pakistan's economy, and a variety of ruminants (including sheep and goats) are reared for the increasing demand of milk, meat and hide products. Haemoparasitic illnesses such as

Anaplasmosis, caused by intracellular gram-negative bacteria Anaplasma marginale is one of the most frequently reported tick-borne disease (TBDs) in tropical and sub-tropical countries, including Pakistan. In the present study, a total of 428 cattle blood samples were collected to examine the prevalence and phylogenetic origin of A. marginale in two important livestock regions of Punjab Province in Pakistan, i.e. Lodhran and Dera Ghazi Khan Districts. In addition, association between occurrence of A. marginale in cattle blood and selected epidemiological factors has been also investigated. The presence of A. marginale genetic material was confirmed in 9% of the tested blood samples taken from cattle in Lodhran and in 17% from Dera Ghazi Khan. Prevalence of A. marginale was significantly higher in cattle from Dera Ghazi Khan. All the cattle breeds from both districts were equally susceptible to A. marginale infection. We reported higher prevalence of A. marginale in cattle living indoors or with other dairy animals in Dera Ghazi Khan district. However, no such relationship was observed in the Lodhran district. Sequencing of the msp1b gene shows 96-99% similarity of A. marginale in the study area to those reported from other parts of Pakistan, South Africa, and Israel. We recommend that large scale tick and tick-borne disease control strategies must be implemented in both districts.

Anaplasma centrale (A. centrale) is an obligate red blood cell residing tick transmitted rickettsiae that has not been studied extensively for its prevalence in cattle along with its epidemiology. Aim of this investigation was to report the seasonal prevalence, phylogeny and epidemiological parameters associated with the prevalence of A. centrale in cattle breeds enrolled from District Layyah in Punjab, Pakistan. A total of 844 blood samples [Cross breed = 300, Holstein Friesian = 244, Sahiwal breed = 300)] were collected from apparently healthy cattle along with epidemiological data during 2017–18. PCR amplified 426 base pair fragment from 16S rRNA gene of A. centrale in 14.4% (122/844) of cattle. Amplified 16S rRNA partial gene sequence of A. centrale were confirmed by DNA sequencing and deposited to GenBank. Highest A. centrale prevalence was observed in spring (24%) followed by autumn (12.4%) summer (10%) and winter (7.1%) seasons. Sahiwal breed (18.3%) was most susceptible to A. centrale infection followed by cross (12.3%) and Holstein Friesian breed (12.3%). 69/844 (8.2%) of Giemsa stained cattle blood smears were also found positive for Anaplasma spp. Farms where animal use to drink pool water and farms where dogs and other dairy animals were living with cattle had higher A. centrale prevalence. Female cattle and dogs having tick burden were found associated with A. centrale infection. Hematological profile was severely disturbed in A. centrale positive cattle. It is recommended that A. centrale should be screened in cattle, in addition to A. marginale, for the effective control of tick born diseases in Pakistan.

Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks (DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. However, few studies have focused on meiotic DSB formation in human reproduction. Here, we report four infertile siblings born to a consanguineous marriage, with three brothers suffering from non-obstructive azoospermia and one sister suffering from unexplained infertility with normal menstrual cycles and normal ovary sizes with follicular activity. An autosomal recessive mutation in TOP6BL was found co-segregating with infertility in this family. Investigation of one male patient revealed failure in programmed meiotic DSB formation and meiotic arrest prior to pachytene stage of prophase I. Mouse models carrying similar mutations to that in patients recapitulated the spermatogenic abnormalities of the patient. Pathogenicity of the mutation in the female patient was supported by observations in mice that meiotic programmed DSBs failed to form in mutant oocytes and oocyte maturation failure due to absence of meiotic recombination. Our study thus illustrates the phenotypical characteristics and the genotype-phenotype correlations of meiotic DSB formation failure in humans.

Anaplasma marginale, Theileria ovis and Theileria lestoquardi are intracellular pathogens that infect a wide variety of animals and cause enormous economic losses worldwide. The present investigation aims to report the occurrence and the phylogeny of these pathogens infecting sheep (N = 330) from Rajanpur District in Punjab (Pakistan) by using msp1b gene for A. marginale and 18 S rRNA for T. ovis and T. lestoquardi. Results revealed that prevalence rates of A. marginale, T. ovis and T. lestoquardi were 7.3%, 6.1% and 1.2%, respectively. Four (1.2%) and one (0.3%) sheep were found to be co-infected with two and three pathogens, respectively. Risk factor analysis revealed that rams were more infected with A. marginale compared to ewes (P = 0.015). In addition, it was observed that animals located in herds having dogs and those in small herds were found more infected, respectively, with T. ovis (P = 0.001) and T. lestoquardi (P = 0.009). Phylogenetic analysis revealed that Pakistani isolates of each detected pathogens clustered together and were closely related to other Pakistani isolates and those from worldwide countries such as Iran, Iraq, Turkey, Sudan, Tanzania, South Africa, and USA. This is the first molecular study reporting the natural infection of Pakistani sheep with these three pathogens. These data need to be taken into account in order to improve the productivity of the livestock sector which is one of main sources of income in the country.

Raising small ruminants is the main source of income for farmers in Pakistan. Economic losses caused by Toxoplasma gondii to small ruminants have been reported worldwide, however reports on molecular detection of T. gondii are lacking in Pakistan despite a large goat population. The current study was carried out from March 2019 till February 2020 to report the seasonal and molecular prevalence of T. gondii in different breeds of goats located in Khanewal district of Punjab, Pakistan. A total of 898 blood goat samples were collected during the four seasons and screened for T. gondii DNA by using PCR based on the amplification of ITS-1 partial sequence. Out of 898 goats, 48 (5.3%) were found positive to T. gondii. The prevalence of T. gondii varied according to season (Chi square test,P = 0.016) and the highest prevalence was observed in goats tested during the summer (8.8%) followed by the spring (5.7%), the winter (4.4%) and the autumn season (2.2%). PCR products positive to T. gondii were confirmed by DNA sequencing and BLAST analysis. Phylogenetic study based on ITS 1 gene provided evidence that the amplified isolates of T. gondii were highly conserved in Pakistani goats. Buck (Fischer exact test, P = 0.002) and farms containing other dairy animals next to goats (Fischer exact test, P = 0.001) and farms with a water supply from pools (Fischer exact test, P = 0.001) were more infected with T. gondii. Infected goats had a reduction on red blood cell count (Two-sample t test, P = 0.01) and hemoglobin concentration (Two-sample t test, P = 0.03) and an increase in the number of monocytes (%) (Two-sample t test, P = 0.05), mean cell hemoglobin (Two-sample t test, P = 0.01) and serum creatinine (Two-sample t test, P = 0.01) as compared to T. gondii uninfected goats. In conclusion, we report a relatively low PCR based prevalence of T. gondii in goats from Khanewal district as previously the serum ELISA test based prevalence of T. gondii in Pakistani goats varied between 19-52%. Control measures should be taken to eradicate T. gondii infection in goats of the study area.

Diafenthiuron is a thiourea compound that has a novel mode of action as it inhibits mitochondrial functioning in insect pests. It has been reported in local newspapers that this pesticide is entering in our fresh water bodies on regular basis and it is a potential threat for aquatic life. The present study was designed to determine effect of Diafenthiuron, a commonly used pesticide in Pakistan, on the hematology, serum biochemical profile and elemental composition of a non-target organism, Labeo rohita (L. rohita). A sub-lethal dose (0.0075 mg L-1) of Diafenthiuron was applied under short (2, 4 and 8 days) and long term (16, 32 and 64 days) experimental conditions. Our results indicated that the pesticide exposed fish had significantly higher white blood count, lymphocyte, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, red cell distribution width than the control group. However, platelets count, plateletcrit and platelet distribution width were significantly reduced in Diafenthiuron treatments than their respective control groups. Concentration of total serum proteins, albumin, globulin, cholesterol, triglycerides and AST were disturbed in pesticide exposed treatments compared to control groups. Comparison of elemental concentrations revealed that calcium, potassium and cadmium concentration varied significantly when compared between Diafenthiuron treated and untreated L. rohita. In conclusion, we are reporting that Diafenthiuron can adversely affect the hematological, serum and elemental concentrations of a non-target organism like L. rohita and may therefore pose a threat to the food web.

Babesiosis, theileriosis and anaplasmosis are among the most commonly reported tick-borne diseases in cattle and are associated with significant economic losses. Through the present study the researchers aimed to report the presence of various pathogens that cause babesiosis, theileriosis and anaplasmosis in cattle collected from different provinces in Saudi Arabia and to report their phylogenetic relationship. A total of 362 blood samples of cattle along with ticks that were present on the cattle were collected from four regions (Riyadh, Al-Kharj, Al-Hasa and Al-Qassim) of Saudi Arabia. Blood samples were screened by polymerase chain reaction (PCR) for the presence of various Babesia, Theileria and Anaplasma species by amplification of their 18S rRNA and/or 23S rRNA genes. A total of 541 ticks were collected and identified from the cattle. These included Hyalomma anatolicum, Hyalomma dromedarii, Hyalomma impeltatum, Hyalomma excavatum, Rhipicephalus annulatus and Rhipicephalus turanicus. Regarding tick-borne pathogens, the overall prevalence was 1.9 % (7/362) for Theileria annulata, (2/362) 0.6 % for Theileria and (21/362) 5.8 % for Anaplasma ovis. Four of the cattle were found to be co-infected with more than one pathogen (1.1 %). We did not detect any Babesia species in the blood of the studied cattle. Prevalence of the Theileria and Anaplasma species was highest in cattle that resided in Riyadh, followed by cattle from Al-Hasa and Al-Qassim. Representative amplified partial-gene sequences of T. annulata (GenBank accession numbers MK826137-39) and A. ovis (GenBank acc. no. MK 880224) were submitted to GenBank. The presence of ticks on cattle was found to be associated with a high prevalence of Theileria spp. (P = 0.02) and Anaplasma ovis (P < 0.001). We report novel genotypes of T. annulata and A. ovis from cattle in Saudi Arabia and we recommend that molecular surveys are undertaken throughout the country to address the prevalence and geographical distribution of tick-borne infections for their effective diagnosis and treatment.

Dwarfism or short stature is a condition when human height is less than 147 cm in adult form. We enrolled two families with multiple siblings suffering from dwarfism and Whole exome sequencing (WES) followed by Sanger sequencing approach was used to report genetic base of their dwarfism. Two novel missense mutations, c.2407C > A, p.Pro803Thr in FGFR1 gene in family one and c.1779C > G, p.F539L in FGFR3 gene in second family were identified as the genetic cause of their phenotype. Sanger sequencing confirmed these mutations in all enrolled patients and mutations followed autosomal dominant mode of inheritance. Multiple sequence alignment by Clustal Omega revealed that domains of FGFR1 and FGFR3 containing mutations are highly conserved. Computational 3D structure generations through homology modeling approach revealed no observable differences in FGFR1 and FGFR3 protein structures when compared between control and patients indicating that observed mutations are responsible for functional changes in proteins.

Creatine is known to rescue animals following brain damage. Present study was designed to demonstrate the effect of long term (15 week) supplementation of 2% creatine monohydrate (Cr), following neonatal hypoxic ischemic insult, on learning and memory formation in male albino mouse. Albino mice pups were subjected to right common carotid artery ligation followed by 8% hypoxia for 25 minutes. Following weaning, animals were separated and grouped on the basis of dietry supplementation for 15 weeks followed by a battery of neurological tests including Morris water maze, open field and rota rod. It was observed that HI mice fed on 2% Cr for 15 weeks performed better than their littermates mice on normal rodent diet during water maze (learning and memory) and rotating rod (neuro-muscular coordination and balance) test while the results of open field test remained unaffected. It was also observed that Cr treated animals had a reduced brain infarct volume than untreated but this difference did not reached statistical significance. We have also observed an overall increase in body weight in Cr treated mice during the study. Over all our results are indicating that long term Cr supplementation is beneficial for male albino following hypoxic ischemic insult.

Currently there are no uniform standard treatments for newborn suffering from cerebral hypoxia-ischemia (HI) and to find new and effective strategies for treating the HI injury remains a key direction for future research. Present study was designed to demonstrate that optimal dose (1 or 3%) of creatine monohydrate (Cr) for the treatment of neonatal HI in female albino mice. On postnatal day 10, animals were subjected to left carotid artery ligation followed by 8% hypoxia for 25 minutes. Following weaning on postnatal day 20, mice were divided into three treatments on the basis of diet supplementation (Normal rodent diet, 1% and 3% creatine supplemented diet) for 10 week. A battery of neurological tests (Rota rod, open field and Morris water maze) was used to demonstrate effect of Cr supplementation on neurofunction and infarct size following HI. Open field test results indicated that Cr supplementation had significantly improved locomotory and exploratory behavior in subjects. It was observed that Cr treated mice showed better neuromuscular coordination (rota rod) and improved spatial memory (Morris Water Maze test). A significant affect of creatine supplementation in reducing infarct size was also observed. Post hoc analysis of post hoc multiple comparisons revealed that mice supplemented with 3% Cr for 10 weeks performed better during Morris water maze test while 1% Cr supplementation improved the exploratory behavior and gain in body weight than control group indicating that Cr supplementation has the potential to improve the neurofunction following neonatal brain damage. This article is part of a Special Issue entitled SI: Brain and Memory.

Purpose Fanconi anemia (FA) genes play important roles in spermatogenesis. In mice, disruption of Fancm impairs male fertility and testicular integrity, but whether FANCM pathogenic variants (PV) similarly affect fertility in men is unknown. Here we characterize a Pakistani family having three infertile brothers, two manifesting oligoasthenospermia and one exhibiting azoospermia, born to first-cousin parents. A homozygous PV in FANCM (c.1946_1958del, p.P648Lfs*16) was found cosegregating with male infertility. Our objective is to validate that FANCM p.P648Lfs*16 is the PV causing infertility in this family. Methods Exome and Sanger sequencing were used for PV screening. DNA interstrand crosslink (ICL) sensitivity was assessed in lymphocytes from patients. A mouse model carrying a PV nearly equivalent to that in the patients (FancmΔC/ΔC) was generated, followed by functional analysis in spermatogenesis. Results The loss-of-function FANCM PV increased ICL sensitivity in lymphocytes of patients and FancmΔC/ΔC spermatogonia. Adult FancmΔC/ΔC mice showed spermatogenic failure, with germ cell loss in 50.2% of testicular tubules and round-spermatid maturation arrest in 43.5% of tubules. In addition, neither bone marrow failure nor cancer/tumor was detected in all the patients or adult FancmΔC/ΔC mice. Conclusion These findings revealed male infertility to be a novel phenotype of human patients with a biallelic FANCM PV.