Extracellular vesicles and exosome: insight from physiological regulatory perspectives

The current study highlights prospective mechanisms of biogenesis of extracellular vesicles (EVs) and potential involvement in cellular signaling and transport with great emphasis to illustrate their role as biomarkers in certain pathologies. The current review highlights EVs, the heterogeneous entities secreted by cells in highly conserved manner. A series of consensus terminologies for various types is yet to be organized. Exosomes, microvesicles and apoptotic bodies are major populations among EVs. EVs are key regulators in cellular physiological homeostasis, disease progression and evolve either from plasma membrane (microvesicles) or fusion of endosomes with exosomes. However, how vesicular inclusions elicit a plethora of biological responses is still not much clear. However, how these vesicular inclusions get packaged and delivered by these EVs shows great involvement in inter- and intracellular cellular signaling and channeling of multiple proteins, variety of RNAs and certain fat molecules. It’s worth to mention that EVs carry small non-coding RNAs (snRNAs) which are involved in multiple cellular molecular events at targeted sites. Moreover, snRNA trafficking through exosomes and microvesicles depicts remarkable potential as non-invasive biomarkers in different clinical disorders especially immune system pathologies, cardiovascular issues, and metabolic syndromes. (AU)

Extracellular vesicles and exosome: insight from physiological regulatory perspectives.

The current study highlights prospective mechanisms of biogenesis of extracellular vesicles (EVs) and potential involvement in cellular signaling and transport with great emphasis to illustrate their role as biomarkers in certain pathologies. The current review highlights EVs, the heterogeneous entities secreted by cells in highly conserved manner. A series of consensus terminologies for various types is yet to be organized. Exosomes, microvesicles and apoptotic bodies are major populations among EVs. EVs are key regulators in cellular physiological homeostasis, disease progression and evolve either from plasma membrane (microvesicles) or fusion of endosomes with exosomes. However, how vesicular inclusions elicit a plethora of biological responses is still not much clear. However, how these vesicular inclusions get packaged and delivered by these EVs shows great involvement in inter- and intracellular cellular signaling and channeling of multiple proteins, variety of RNAs and certain fat molecules. It's worth to mention that EVs carry small non-coding RNAs (snRNAs) which are involved in multiple cellular molecular events at targeted sites. Moreover, snRNA trafficking through exosomes and microvesicles depicts remarkable potential as non-invasive biomarkers in different clinical disorders especially immune system pathologies, cardiovascular issues, and metabolic syndromes.

Association of life style and dietary habits with blood choline and cardiovascular outcome.

Whole-blood choline, plasma choline and serum choline are emerging biomarkers in cardiovascular diseases (CVD). To examine the association of Whole-blood choline is an early predictor for cardiac events. In case control study, we enrolled 240 individuals including 120 normal (39 females and 82 males) and 120 cases (49 females and 71 males) where age limit was >40 years) Information through interviews, family disease history, 24 recall diet assessment and blood sampling. Odds ratios express the associated risks with CVD and without CVD patients. In healthy populations, good dietary habits and active lifestyle were observed. The number of participants with CVD were smokers than normal. In men, and women the risk was observed highly significant. (p=0.0049) Different blood parameters like Triglycerides, Uric Acid, Urea, Creatinine, CRP and ESR were non-significant observed. In females the low carbohydrates and high protein and frequent salad vegetable consumption observed. On the other hand, men consume more carbohydrates. Body mass index was significantly with p= 0.036 (OD 1.12 95% 1.00-1.26). The total fats (p=0.017) (OD 1.3301 95% 1.05-1.69) total carbohydrate (p=0.076) (OD 1.1536 95% 0.98-1.35) and total proteins (p=0.287) (OD 1.1456 95% 0.89-1.47) effecting respectively.  The Blood choline level was significant observed between cases (p=0.026) OD (0.944 95%0.89- 0.99).

Genetic Diversity of Canine Babesia Species Prevalent in Pet Dogs of Punjab, Pakistan.

Canine babesiosis is a serious threat to dogs' health worldwide, caused by the intra-erythrocytic Babesia species. The present study was carried out in pet dogs presented at three clinics of Lahore and one of Narowal in Punjab, Pakistan. Two hundred blood samples (50 from each clinic) were collected and screened by microscopy for Babesia spp. Out of 200 samples, 84 (42%) were found to be positive for babesiosis. The highest number of positive cases (50%) was recorded in dogs at Narowal clinic. Non-significant variation (p > 0.05) was observed in the prevalence of babesiosis in dogs in relation to sex and age. Positive samples were further confirmed by a polymerase chain reaction using 18S-rRNA genus-specific and species-specific primers. Amplicons were further analyzed by nucleotide sequencing for genetic diversity. Babesia canis and gibsoni were confirmed by genome sequencing in all diseased dogs. These isolates closely resembled each other, but differed from previous reported strains. In conclusion, pet dogs suffering from babesiosis were infected with B. canis and gibsoni, while in other countries, other Babesia species are also prevalent.

Molecular Epidemiology and Prevalence of Theileria lestoquardi and Theileria ovis Infection in Goats Infested With Tick Vectors From Multan, Pakistan.

Caprine theileriosis is a major production problem in regions of the world that rely on goats as a major source of milk, meat, and other means of economic income. Theileria ovis and Theileria lestoquardi are responsible for caprine theileriosis in Pakistan. The present study examined the prevalence of Theileria spp. infection using polymerase chain reaction (PCR) and light microscopy of blood collected from goats from Multan, Pakistan. In addition, the prevalence and identity of the genus of ticks feeding on these goats was determined on specimens collected at the time of blood sampling. The current project hypothesized that Theileria spp. prevalence would be higher in goats infested with ticks than goats without tick infestation. Four hundred and sixty-three blood samples from goats were analyzed, and the prevalence of infection was 16.0% (95% confidence interval [CI] = ±5.96) 74 of 463 by PCR and 5.4% (95% CI = ±7.79) 25 of 463 by microscopic examination. Thirty-six (48.6%, 95% CI = ±4.96) and 30 (40.5%, 95% CI = ±7.10) of 74 samples were positive by PCR for T. ovis and T. lestoquardi, respectively. Eight samples (10.0%, 95% CI = ±9.61) had mixed infections of these Theileria species. Infection was observed significantly (P < 0.05) more often in male 22.8%, 95% CI = ±3.85 (23/101) than in female 14%, 95% CI = ±2.36 (51/362) goats. No statistical (P > 0.05) difference in prevalence was present among the three age groups of ≤1 yr (12.6%, 95% CI = ±6.70), 1-4 yr (16.2%, 95% CI = ±8.24), and ≥4 yr (17.7%, 95% CI = ±11.30) goats examined. Two hundred and one (43.4%, 95% CI = ±7.45) goats were infested with ticks, Rhipicephalus spp. and Haemaphysalis spp., which are capable of transmitting Theileria spp. Tick infestation was found to be a significant (P < 0.05) risk factor for infection. Prevalence of Theileria infection in goats was significantly higher (P < 0.05) in the summer compared with the other three seasons. Data from this study support our hypothesis and demonstrate that both T. ovis and T. lestoquardi are highly prevalent in goats from Multan, Pakistan.

Prevalence and risk factors for IgG antibodies to Neospora spp. in three types of equids from Southern Punjab, Pakistan.

Equine hosts suffer from neurological disease, congenital infection, and reproductive problems associated with Neospora spp. infection. We conducted a cross sectional study using sera from 631 equids (324 horses, 218 donkeys and 89 mules) from the southern region of Punjab province, Pakistan to determine the prevalence of antibodies against Neospora spp. in this diverse group of equines. Fisk factors associated with seropositivity were evaluated statistically based on equine type, breed, age, husbandry, breeding methods, and reproductive history. Prevalence of antibodies to Neospora spp. was detected using a commercially available competitive ELISA kit. We detected IgG antibodies to Neospora spp. in 23.3% of the equids with prevalence by host being 16.0% in horses, 32.6% in donkeys and 26.9% in 89 mules. Statistically significant (P < 0.05) differences in prevalence were observed among these hosts. Prevalence ranged in breeds of horses from 3.7% in the Morna breed, 12.4% in breeding stock, 26.8% in draughting stock, to 31.4% in unknown breeds. No significant (P > 0.05) difference in prevalence was noted among age groups. Prevalence was significantly (P < 0.05) higher in female equines with a history of abortion than those with no history of abortion. This is the first report documenting Neospora spp. in equines from Pakistan and it provides evidence that Neospora spp. is associated with abortions in female equines in Pakistan.

Prevalence of Toxoplasma gondii, Neospora caninum, and Sarcocystis Species DNA in the Heart and Breast Muscles of Rock Pigeons (Columbia livia).

Little is known about the prevalence of protozoan parasites in the muscles of rock pigeons (Columbia livia). The muscles from 54 (heart from 45 and breast from 54) rock pigeons were examined for DNA of Toxoplasma gondii, Neospora caninum, and Sarcocystis species using PCR. Twenty-four were female and 30 were males. The birds were part of flocks of pigeons housed at the tombs of saints in Lahore, Pakistan. Birds that died or were euthanized due to poor health were submitted for necropsy at the Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan, where DNA isolations and PCR were conducted. Nineteen (35.1%) of the birds were positive for T. gondii DNA. Seven males and 12 females were positive. Breast tissue was always infected in T. gondii positive birds, while the heart was infected in 13 (28.8%) of breast positive birds. Five (9.2%) of the pigeons, 2 males and 3 females, were positive for N. caninum. The distribution of N. caninum DNA was more variable in the muscles of pigeons than T. gondii and was found only in the heart of 1 (female), heart and breast muscle of 2 (male), and only the breast muscle of 2 birds (female). One of the 54 rock pigeons (female) was positive for both T. gondii (heart and breast) and N. caninum (heart only). Two of the positive Neospora caninum amplicons were sequenced and had 97% nucleotide identity with N. caninum isolates. Sarcocystis DNA was not found in any bird. The prevalence of T. gondii in rock pigeons and their predation by cats suggest that they may play an unrecognized role in maintaining environmental contamination with T. gondii oocysts by cats. Our study indicates that rock pigeons are intermediate hosts of N. caninum and this information will aid in understanding the epidemiology of N. caninum.

Cutaneous Leishmaniasis in the Metropolitan City of Multan, Pakistan, a Neglected Tropical Disease.

Cutaneous leishmaniasis is a vector-borne infection caused by flagellated parasitic protozoans in the genus Leishmania. Promastigotes are the infective stage, which are transmitted by the bite of female sand flies of the genera Phlebotomus or Lutzomyia. Three clinical forms of Leishmania infection are recognized in humans, and they are caused by different Leishmania species. They are cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (ML), and visceral leishmaniasis (VL). Up to 2 million cases of leishmaniasis are reported annually worldwide. In Pakistan, CL is widely distributed in endemic areas. In the current study, a survey was conducted to investigate the occurrence of CL in an endemic area of the metropolitan city of Multan, Pakistan. The material for diagnosis of CL was obtained from 461 patients from 239 clinical laboratories and health care centers in Multan. Of these patients, 42.9% (198 of 461) having positive findings of amastigotes in Giemsa stained tissue impression smears. The distribution of CL skin lesions was highly variable. Most patients (76.2%) had a single lesion. Most CL lesions were found on a single hand (23.7%). The prevalence of two CL lesions per patient was 21.7%, while three or more positive CL lesions were observed in 2.0% of patients. The 198 CL-positive patients ranged in age 10-40 yr old, 101 were males, and 97 were females. Logistic regression analysis of leishmaniasis versus age and gender showed that both the age and gender had a significant (P < 0.05) effect on the occurrence of infection. Outbreaks of CL were reported in 14 of the 68 Union Councils of Multan.

Prevalence of antibodies to Neospora caninum in the serum of camels (Camelus dromedarius) from central Punjab, Pakistan.

Infection with the Apicomplexan parasite Neospora caninum occurs in domestic and wild animals worldwide. This parasite causes significant economic losses in the dairy industry worldwide. The current study was designed to assess the prevalence of N. caninum antibodies in the sera of camels (Camelus dromedarius) from Rakh Mahni, Bhakkar, Pakistan, to better understand the epidemiology of this important cause of bovine abortion in Pakistan. Eighty-one sera samples collected from apparently healthy camels (male = 19, female = 62) were tested for antibodies against N. caninum by using a commercially available competitive Enzyme Linked Immunosorbent assay diagnostic kit. Nine (11.1%) of 81 sera samples were found positive for antibodies to N. caninum. No significant (P > 0.05) difference in seroprevalence was observed between male and female camels or between different breeds. An increasing trend of seropositivity to N. caninum was seen with the age of animals indicating postnatal exposure to N. caninum infection. However, this increase in prevalence was not significant (P > 0.05). However, a significant (P < 0.05) difference in seroprevalence was noted among feeding style of animals, i.e., stall feeding, grazing, and mixed (grazing and stall feeding) feeding style. The prevalence was significantly (P < 0.05) higher in female animals 26.1% (6/23) with abortion history that of animals have had no such history 5.1% (2/39). Presence of antibodies to N. caninum in the sera of camels indicates that further studies are required to evaluate the importance of N. caninum as a cause of abortion or neonatal disease in these animals.

Entamoeba infections in different populations of dogs in an endemic area of Lahore, Pakistan.

Entamoeba histolytica, a protozoan parasite that affects humans and other primates all over the world. It is a common waterborne pathogen in endemic areas that have fecal oral transmission cycle. The aim of the present study was to examine the prevalence of E. histolytica and other Entamoeba species cysts in three different dog populations. Fecal samples from 600 dogs were collected and processed to detect Entamoeba cysts using the triple fecal test (light microscopy) and fecal antigens of E. histolytica were detected using a fecal antigen ELISA (TechLab E. histolytica II). Because it is impossible to differentiate E. histolytica from Entamoeba dispar and E. moshkovskii, using light microscopy we referred to all cysts morphologically consistent with E. histolytica as E. histolytica/dispar/moskovskii to reflect this uncertainty. Samples from 197 household dogs without clinical signs, 122 samples from household dogs exhibiting clinical signs of diarrhea, dysentery and vomiting and 281 stray dogs with no specific clinical signs were examined. Entamoeba histolytica-like cysts were observed in 94 (15.6%, 95% CI=±3.88) by triple fecal test microscopy and E. histolytica antigens were demonstrated in 66 (11%, 95% CI=±4.41) by fecal antigen ELISA in 600 fecal samples. Significant differences (P≤0.05) in prevalence were found between the three populations. Twenty (10.1%, 95% CI=±7.86) and 11 (5.6%, 95% CI=±7.70) of 197 fecal samples from household dogs without clinical signs were positive by microscopy and by antigen ELISA, respectively. Twenty-nine (23.8%, 95% CI=±6.58) and 23 (18.8%, 95% CI=±7.81) of 122 the fecal samples from household dogs with clinical signs were positive by microscopy and by antigen ELISA, respectively. Forty-five (16.01%, 95% CI=±5.62) and 32 (11.3%, 95% CI=±6.38) of 281 fecal samples from stray dogs were positive by microscopy and by fecal antigen ELISA, respectively. Dogs from the youngest age group (6 months to 1 year) were more likely to be E. histolytica antigen positive than were dogs from the other two older age groups, with a significant difference (P≤0.05) between all age groups. Statistically, no significant (P≥0.05) difference of prevalence was seen in male and female dogs. The local dogs had the highest prevalence rate of E. histolytica antigens (36 of 246, 14.2%, 95% CI=±6.32) followed by imported breeds (11 of 115, 9.5%, 95% CI=±10.4) and crossbred (19 of 239, 8.3%, 95% CI=±7.47), indicating a significant (P≤0.05) trend of positivity between various breeds of dogs. These findings suggest that dogs may play an important role in the epidemiology of this pathogen.

Prevalence of Entamoeba histolytica -like cysts compared to E. histolytica antigens detected by ELISA in the stools of 600 patients from three socioeconomic communities in the Metropolitan City of Lahore, Pakistan.

Amoebiasis, caused by Entamoeba histolytica , has a worldwide distribution and is of public health significance in many developing countries. It has a fecal-oral transmission cycle and is most prevalent in developing countries in regions where substandard sanitary conditions exist due to poverty. Little is known about the epidemiology of E. histolytica infection and its presence in different socioeconomic communities in developing countries. We undertook the present study in the city of Lahore, Pakistan, and our prediction was that the prevalence of E. histolytica -like cysts and E. histolytica stool antigen would be lower in patients from upper socioeconomic levels than in individuals from middle or lower socioeconomic levels. We investigated the prevalence of E. histolytica in humans from 3 socioeconomic communities in territories of Lahore, Pakistan. Six hundred fecal samples were collected and examined using both microscopy (triple fecal test) to detect cysts of E. histolytica -like amoeba and ELISA (stool antigen ELISA) to demonstrate diagnostic stool antigens of E. histolytica . Samples were from individuals living under conditions deemed to be upper socioeconomic class (n = 287), middle socioeconomic class (n = 172), and lower socioeconomic class (n = 141). The total prevalence of positive samples was 22.5% (135/600) by triple test and 16.8% (101/600) by stool antigen ELISA in the 600 fecal samples. Statistically, significant (P < 0.05) differences in prevalence were seen between the 3 socioeconomic class groups. Forty-four (15.3%) and 32 (11.1%) of 287 in the fecal samples from the upper socioeconomic class were positive by triple test and by antigen ELISA, respectively. Thirty-nine (22.6%) and 29 (16.8%) of 172 in the fecal samples from the middle socioeconomic class were positive by the triple test and by antigen ELISA, respectively. Fifty-two (36.8%) and 40 (28.3%) of 141 in the fecal samples from the lower socioeconomic class were positive by the triple test and by antigen ELISA, respectively. We accept our hypothesis based on these findings. We also demonstrated that fecal samples collected from the youngest age group (1 mo-5 yr) were more likely to be positive for E. histolytica antigens than were samples from the other 3 age groups, and that prevalence was significantly higher (P < 0.05) in the summer than in the other 3 seasons. These results highlight the importance of surveillance of this relatively ignored pathogen in this developing metropolitan city in Pakistan.

Neospora caninum prevalence in dogs raised under different living conditions.

Neospora caninum is an important cause of abortion in dairy cattle worldwide. Dogs are important in the epidemiology of N. caninum because they act as definitive hosts shedding oocysts in the environment. Vertical transmission of the parasite is well recognized as an important aspect of the epidemiology of the parasite but the importance of horizontal transmission has been less studied. A N. caninum competitive ELISA was used to examine serum samples from 600 dogs that were raised under 4 different living conditions. Samples from 138 dogs living on 24 dairies with a prevalence (0-70%) of anti-N. caninum antibodies in the cattle, 294 pet dogs without neurological signs, 76 from pet dogs exhibiting neurological signs, and 92 stray dogs were examined. The overall seroprevalence of N. caninum was 23.5% (95% CI = ± 2.99) in the 600 dogs. Significant (P < 0.05) differences were observed between the 4 different populations of dogs. The number of N. caninum positive samples were: 51 (36.9%, 95% CI = ± 3.09) of 138 dogs from dairies, 31 (10.5%, 95% CI = ± 6.38) of 294 pet dogs without neurological signs, disorders, 22 (28.9%, 95% CI = ± 6.70) of 76 pet dogs with neurological signs, and 37 (40.2%, 95% CI = ± 2.83) of 92 stray dogs. Seropositivity to N. caninum in dogs from dairies was associated with a high prevalence of N. caninum antibodies in the cattle. At the 3 dairies where no dogs were present, the seroprevalence to N. caninum in the cattle was significantly lower (P < 0.05) than in the 21 dairies where dogs were present. Seroprevalence was significantly higher (P < 0.05) in male dogs (97 of 366; 26.5%, 95% CI = ± 3.40) than in female dogs (44 of 234; 18.8%, 95% CI = ± 5.65). Seroprevalence in dogs increased with age suggesting postnatal exposure to N. caninum infection however, this increase was not significant (P > 0.05). The prevalence of N. caninum antibodies was not significantly (P>0.05) different in dogs based on breed. These findings suggest a relationship between N. caninum infection of dogs from dairies and cattle on these dairies. However, further research is required to determine what is the most important way dogs acquire infection and how to prevent dogs from shedding oocysts.

Effects of age and breed on the prevalence of Neospora caninum in commercial dairy cattle from Pakistan.

Neospora caninum is a major cause of bovine abortion worldwide. A serological survey was carried out to determine the seroprevalence of exposure to N. caninum in dairy cattle based on age and breed from Punjab and Sindh provinces, Pakistan. Serum samples from 641 animals from 12 herds from Punjab (n = 7) and Sindh (n = 5) provinces were tested for antibodies against N. caninum using a commercially available competitive enzyme-linked immunosorbent assay. Positive reactions to N. caninum were seen in 277 (43%) of the 641 of the samples. Seropositive animals were present in all 12 herds. Animals over 2 yr of age (47%) and crossbreds (55%) were more likely to be seropositive than the other cattle examined. These results indicate that N. caninum infection is widespread among dairy cattle in Pakistan.

Seroprevalence of Neospora caninum and Brucella abortus in dairy cattle herds with high abortion rates.

The protozoan Neospora caninum and the bacterium Brucella abortus are well-recognized causes of abortion in dairy cattle. Serum samples (n  =  240) from aborting (n  =  141) and at-risk (n  =  99) animals from 5 herds with high abortion rates in Punjab Province, Pakistan, were tested for antibodies to N. caninum using monoclonal antibody-based ELISA and for antibodies to B. abortus using the serum agglutination test. Antibodies to N. caninum and B. abortus were detected in 105 (43.8%) and 135 (56.3%) cattle, respectively. Prevalences of antibodies to N. caninum and B. abortus were higher in aborting cows (46.8% and 76.6%, P < 0.05) than in animals at risk (39.4% and 27.3%, P > 0.05). Sixty-six animals (27.5%) were seropositive to both N. caninum and B. abortus , and results showed no significant difference (P > 0.05) with respect to geographical district, breed, and age. This is the first report of N. caninum infection among dairy cattle herds in Pakistan.