High Efficacy of Green Synthesized Silver Nanoparticles for Treatment of Toxoplasma Gondii Infection Through Their Immunomodulatory, Anti-Inflammatory, and Antioxidant Potency.

The present experimental survey designed to green synthesis, characterization, as well as in vitro and in vivo anti-Toxplasma gondii activity of silver nanoparticles (SLN) green synthesized by Lupinus arcticus extract. SLN were green synthesized based on the reducing by L. arcticus extract through the precipitation technique. In vitro lethal effects of SLN on T. gondii tachyzoites, infectivity rate, parasites inside of the human macrophage cells (THP-1 cells), nitric oxide (NO) triggering, and iNOS and interferon gamma (IFN-γ) expression genes were evaluated. In vivo, after establishment of toxoplasmosis in BALB/c mice via T. gondii ME49 strain, mice received SLN at 10 and 20 mg/kg/day alone and combined to pyrimethamine at 5 mg/kg for 14 days. SLN exhibited a spherical form with a size ranging from 25 to 90 nm. The 50% inhibitory concentration (IC50) value of SLN and pyrimethamine against tachyzoites was 29.1 and 25.7 µg/mL, respectively. While, the 50% cytotoxic concentration (CC50) value of SLN and pyrimethamine against THP-1 cells was 412.3 µg/mL and 269.5 µg/mL, respectively. SLN in combined with pyrimethamine obviously (p < 0.05) decreased the number and size of the T. gondii cysts in the infected mice. The level of NO, iNOS and IFN-γ genes was obviously (p < 0.001) upregulated. SLN obviously (p < 0.05) decreased the liver level of oxidative stress and increased the level of antioxidant factors. The findings displayed the promising beneficial effects of SLN mainly in combination with current synthetic drugs against latent T. gondii infection in mice. But we need more experiments to approve these findings, clarifying all possible mechanisms, and its efficiency in clinical phases.

Potent In Vitro and In Vivo Effects of Stachys lavandulifolia Methanolic Extract against Toxoplasma gondii Infection.

The present study aimed to evaluate the in vitro, in vivo, and safety of Stachys lavandulifolia Vahl. methanolic extract (SLME) against acute toxoplasmosis caused by Toxoplasma gondii RH strain in mice. METHODS: MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to evaluate the in vitro effect of the SLME on T. gondii tachyzoites. Totally, 72 male BALB/c mice (40 mice for in vivo evaluation of SLME and 32 mice for its toxicity effects on liver and kidney serum enzymes) were used for the present investigation. At first, 40 mice were orally pre-treated with the SLME at doses of 25, 50, and 75 mg/kg/day for two weeks. Mice were checked daily, and the rate of survival and the mean number of tachyzoites were recorded. Liver lipid peroxidation (LPO) and nitric oxide (NO) levels, the effects on kidney and liver function, as well as the expression level of the proinflammatory cytokines such as interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ), were studied by the quantitative real-time PCR. Flow cytometry analysis was performed on the effects of SLME on the detection of apoptotic and necrotic cells in T. gondii tachyzoites. RESULTS: The SLME at the concentrations 75 and 150 µg/mL completely killed the tachyzoites after 2 hr of incubation. SLME at 25, 50, and 75 mg/kg/day increased the survival rate of infected mice by the sixth, seventh, and eighth days, respectively. SLME also significantly (p < 0.05) decreased the LPO and NO levels and upregulated the IL-1ß and IFN-γ mRNA gene expression levels, whereas no considerable change was observed in the serum level of kidney and liver enzymes. Flow cytometry analysis revealed the prompted early and late apoptosis after exposure to T. gondii tachyzoites with various concentrations of SLME. CONCLUSION: We found the relevant in vitro anti-Toxoplasma effects of SLME against T. gondii. Moreover, the results confirmed the promising in vivo prophylactic effects of SLME. SLME provokes the innate immune system, induces apoptosis, modulates the proinflammatory cytokines, and inhibits hepatic injury in infected mice. With all these descriptions, further surveys are required to support these findings and elucidate this plant's possible mechanisms of action.

Performance of diagnostic assays used to detect Cryptosporidium oocysts in faecal samples of cattle in Kuwait and genotyping of Cryptosporidium species.

BACKGROUD: Cryptosporidium species are zoonotic protozoan parasites responsible for gastroenteritis in various animals and humans. The diagnosis of Cryptosporidium presents many challenges. This research attempted to match the diagnostic efficiency of the modified Ziehl-Neelsen technique (mZN), immunochromatographic assays (IC), and enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium in faecal samples of cattle in Kuwait. In addition, polymerase chain reaction (PCR) was utilised to determine the predominant species infecting cattle in Kuwait and correlating the detected species with the results of different diagnostic tests used, the presence or absence of clinical signs, and the age group of the infected cattle. RESULTS: Of 400 analysed faecal samples, Cryptosporidium positive samples were 23%, 15.25%, and 14% using IC, ELISA, and mZN. IC had the highest sensitivity (74.07%), and mZN had the highest specificity (98.29%) using a composite reference standard (CRS) as a gold standard. The rapid IC test results in high false-positive results of cryptosporidiosis, whereas using mZN alone is insufficient to declare a negative faecal sample. Only 74.5% (35/47) of Cryptosporidium-positive samples by the three assays could be amplified by PCR. This study was the first to genotype Cryptosporidium in Kuwait. Cryptosporidium parvum (n = 26) was the dominant species detected from cattle samples, followed by C. andersoni (n = 6), C. bovis (n = 2), and C. raynae (n = 1). The findings showed a statistically relevant relationship between diarrhoea and the detection of Cryptosporidium spp. in faecal samples of cattle (p-value = 0.0003). Pre-weaned calves were the most vulnerable age group to Cryptosporidium spp. infection (p-value = 0.0007). CONCLUSION: For screening of Cryptosporidium infection in faecal samples, antigen detection or PCR methods combined with one of the microscopy techniques should be used. Cryptosporidium parvum was the prepoderant Cryptosporidium spp. recovered from cattle samples in Kuwait followed by C. andersoni. Cryptosporidium parvum is a significant risk factor for diarrhoea in pre-weaned calves. However, further study is needed as many other causes of diarrhoea in calves must be ruled out before a diagnosis of Cryptosporidium diarrhoea can be made.

Epidemiological and Molecular Study of Cryptosporidium in Preweaned Calves in Kuwait.

Cryptosporidium is a worldwide enteric protozoan parasite that causes gastrointestinal infection in animals, including humans. The most notable species is Cryptosporidium parvum because of its zoonotic importance; it is also the leading cause of cryptosporidiosis in preweaned calves. A cross-sectional study was conducted to determine the prevalence of Cryptosporidium infection, investigate the potential risk factors, and use molecular diagnosis to identify the predominant Cryptosporidium spp. in preweaned calves in Kuwait. Of 175 preweaned calves, Cryptosporidium antigens were detected in 58 (33.1%) using rapid lateral immunochromatography assay (IC). Calves less than one month of age (OR = 4.32, p = 0.0001) and poor hygiene (OR = 2.85, p = 0.0075) were identified as significant risk factors associated with Cryptosporidium infection. Molecular identification revealed that C. parvum (62.8%) was the dominant species infecting preweaned calves in Kuwait. In contrast, C. bovis and C. andersoni were recorded at 5.7% and 2.9%, respectively. All C. parvum gp60 nucleotide sequences were subtype IIaA15G2R1. Calves could be a source of C. parvum infection due to the similarity of the subtypes recorded previously in Kuwaiti children and preweaned calves in this study. Therefore, more research is needed to understand the Cryptosporidium transmission cycle in Kuwait.

Evaluation of immunochromatography test for detection of four enteropathogens in the feces of sheep and goats in Kuwait.

Background: Diarrhea in newborn small ruminants continues to be the cause of significant financial loss in sheep and goat farms worldwide. Commercial immunochromatographic (IC) assays have been designed and evaluated to be used for the diagnosis of diarrhea in cattle; however, there are no trials to use rapid tests in small ruminants. Aim: This study was carried out in Kuwait to evaluate the performance of the rapid immunochromatography test (BoviD-4, BioNote, Inc, Korea) for diagnostics of Cryptosporidium, rotavirus A (RVA), bovine coronavirus (BCoV), and Escherichia coli K99 (E. coli K99) in fecal samples of sheep and goats. Methods: A total of 85 samples were examined using BoviD-4, and the results were compared with that of polymerase chain reaction for Cryptosporidium, RVA, and BCoV, whereas for E. coli K99 it was by isolation and identification as reference tests. Results: The kappa test agreement results between the BoviD-4 and reference tests were 0.870 (perfect), 0.783 (substantial), 0.728 (substantial), and 0.281 (fair) for the detection of E. coli K99, Cryptosporidium, RVA, and BCoV, respectively. The sensitivity of BoviD-4 kit was 91.2%, 80.0%, 90.0%, and 37.5% and the specificity was 88.2%, 96.0%, 96.4%, and 92.2% for Cryptosporidium, RVA, E. coli K99, and BCoV, respectively. Conclusion: The Bovid-4 kit can be used as a rapid pen-side test for Cryptosporidium spp., E. coli K99, and RVA in the field. Nonetheless, care must be taken while interpreting the BCoV results of the kit.

Cross-sectional study and genotyping of rotavirus-A infections in ruminants in Kuwait.

BACKGROUND: Group A rotaviruses (RVA) are zoonotic pathogens responsible for acute enteritis in human and neonatal ruminants. This research aimed to determine the prevalence of RVA in ruminants (cattle, sheep, and goats) and investigate the circulating RVA genotypes in these animals in Kuwait. We conducted a cross-sectional study to detect RVA in ruminants, using an immunochromatography test (IC), direct sandwich ELISA test, and real-time RT-PCR (RT-qPCR) assay using fecal samples. RESULTS: A total of 400 cattle, 334 sheep, and 222 goats were examined. The prevalence of RVA was 5.3, 1.2, and 2.3%, respectively, using IC. The ELISA test detected RVA from 4.3% of cattle, 0.9% of sheep, and 1.8% of goats. There was a significant association between the occurrence of diarrhea and the presence of RVA in bovine fecal samples (p-value = 0.0022), while no statistical association between diarrhea and the presence of RVA in fecal samples of sheep and goats was observed (p-value = 0.7250; p-value = 0.4499, respectively). Twenty-three of the IC-positive samples (17 from cattle, two from sheep, and four from goats) were tested using a RT-qPCR RVA detection assay targeting the NSP3 gene. The results showed that 21 of 23 IC-positive samples tested positive by RT-qPCR. Detection of RVA genotypes revealed that G10P[11] was the predominant strain in cattle (58.8%), followed by G8P[1] (11.7%). One sheep sample was genotyped as G8P[1]. In addition, G6P[1] and G6P[14] were detected in goat samples. CONCLUSION: The present study revealed that the IC was more sensitive in detecting RVA antigen in fecal samples than the ELISA test. A higher occurrence of RVA infection was observed in cattle than in sheep and goats. This study suggests that RVA might be a risk factor of diarrhea in bovine calves less than 2 weeks old. This research also demonstrates the circulation of RVA in sheep and goat populations in Kuwait. Finally, the G10P[11] RVA genotype was the most prevalent genotype identified from cattle samples.

Epidemiological observations on cryptosporidiosis and molecular characterization of Cryptosporidium spp. in sheep and goats in Kuwait.

Molecular epidemiological analysis of cryptosporidiosis in Middle Eastern countries suggests that small ruminants could play a major role in the transmission of Cryptosporidium spp. to humans, with a dominance of Cryptosporidium parvum, especially its IId subtypes. However, little information is available on the epidemiology and risk factors of cryptosporidiosis as well the distribution of Cryptosporidium species/genotypes and subtypes in small ruminants in this area, including Kuwait. In the present study, 47 farms from several areas in Kuwait were visited once during October 2014 to September 2015 to collect data on risk factors associated with Cryptosporidium infection. Fecal samples from 334 sheep and 222 goats were examined for Cryptosporidium oocysts by Ziehl-Neelsen staining (ZN) and antigens by enzymatic immunoassay (EIA). The Cryptosporidium prevalence was higher when samples were examined by EIA than ZN (11.4 and 7.2% in sheep and goats by EIA, compared with 4.2 and 3.6% by ZN, respectively). Young age (less than 3 months) and closed housing system are risk factors of Cryptosporidium infection. A correlation between fecal consistency and the occurrence of Cryptosporidium spp. was observed; non-formed fecal samples were often found positive. Molecular characterization of 30 ovine and caprine samples using PCR-RFLP analysis of the small subunit rRNA gene revealed the presence of C. parvum in 23 samples, Cryptosporidium ubiquitum in five samples, and Cryptosporidium xiaoi in two samples. Sequence analysis of C. parvum at the 60 KDa glycoprotein gene locus identified two subtypes, IIaA15G2R1 and IIdA20G1, with the latter being more common (in 2 and 20 successfully subtyped samples, respectively). Only one subtype of C. ubiquitum (XIIa) was recorded. Cryptosporidiosis in small ruminants apparently poses public health problem in Kuwait.

Study on parasites from farm animals in Kuwait.

No doubt, farm animals are essential as a source of milk, protein, and leather and wool ... etc. But, they are always exposed to ecto- and endo-parasites, which cause diseases conditions that may end in death. This study evaluated farm animal parasitosis. Thus, different animal farms were visited to collect fecal samples and data to determine the infection rates with parasites and the relationship between animal management and parasitism in Kuwait. Out of 86, 17, 20, 96 & 52 cattle, sheep, goats, horses and camels examined, 5.5, 17.5, 10, 9.3 and 2.5% respectively were infected with different parasites. These parasites were Ascarids in cattle and horses, Strongylids in cattle, horses and camels, and Eimeriids in cattle and small ruminants. Eimeria spp. were the most prevalent parasite particularly in small ruminants. The relationship between Eimeria infection and management in small ruminant farms was discussed.

Preliminary study on cryptosporidiosis in livestock from Kuwait.

Out of 225 fecal samples (40 of calves, 128 of lambs and 57 of goat kids) received in the Veterinary Laboratories, Kuwait, 43 (19.1%) were found to be positive for Cryptosporidium using the conventional method, modified acid-fast stain, and the immunologic technique, the immuno-chromatography assay. Calf fecal samples showed the highest infection rate (37.5%), followed by goat kids (21%) and then Lambs (12.5%). Results obtained by conventional method were found to be consistent with those detected using the immunologic technique.

Enteric protozoan parasites in stray cats in Kuwait with special references to toxoplasmosis and risk factors affecting its occurrence.

In Kuwait, stray cats were surveyed for enteric protozoan infection using fecal examination and their sera were tested for Toxoplasma gondii IgG using indirect hemagglutination test (IHAT) as well as for feline immunodeficiency virus (FIV) antibodies and feline leukaemia virus (FeLV) antibodies using ELISA. Out of 240 fecal samples examined 22 (9.2%) were found to be infected with oocysts of four species of coccidian protozoa. Isopspora felis was the most predominant enteric protozoan parasite (7.1%), followed by T. gondii (2.1%), I. rivolta (1.6), Sarcocystis was only found in one case (0.4%). Juvenile cats ( 6 months old) had higher infection rate with oocyst of enteric protozoa than older cats (p-value 0.001). Sero-survey of 240 stray cats revealed that 19.6% were positive to T. gondii IgG. Toxoplasma sero-positivity was observed in higher number of adults compared to young cats suggests that with age the risk of exposure to T. gondii increases. While concurrent retroviral infections were not found to be associated with increased risk for developing T. gondii antibodies.

The decomposition of rabbit carcasses and associated necrophagous Diptera in Kuwait.

Rabbit carcasses were used to compare rates of decomposition and associated assemblages of Diptera at four discernable habitat types in Kuwait; a country of a region with a paucity of such reference data. Carcasses in an urban habitat showed faster decomposition (as measured by percentage weight loss) than in agricultural, coastal or desert habitats, even with accumulated degree days (ADD) as the explanatory variable (t=2.73, df=34, p=0.010) to compensate for temperature differences. Taxa of Diptera at the four habitats became more similar as decomposition progressed, suggesting such differences between habitats were not marked. The occurrence of Chrysomyia megacephala and Lucilia sericata had not previously been recorded in Kuwait.