Blood Cytokine Analysis Suggests That SARS-CoV-2 Infection Results in a Sustained Tumour Promoting Environment in Cancer Patients

Cytokines, chemokines, and (angiogenic) growth factors (CCGs) have been shown to play an intricate role in the progression of both solid and haematological malignancies. Recent studies have shown that SARS-CoV-2 infection leads to a worse outcome in cancer patients, especially in haematological malignancy patients. Here, we investigated how SARS-CoV-2 infection impacts the already altered CCG levels in solid or haematological malignancies, specifically, whether there is a protective effect or rather a potentially higher risk for major COVID-19 complications in cancer patients due to elevated CCGs linked to cancer progression. Serially analysing immune responses with 55 CCGs in cancer patients under active treatment with or without SARS-CoV-2 infection, we first showed that cancer patients without SARS-CoV-2 infection (n = 54) demonstrate elevated levels of 35 CCGs compared to the non-cancer, non-infected control group of health care workers (n = 42). Of the 35 CCGs, 19 were common to both the solid and haematological malignancy groups and comprised previously described cytokines such as IL-6, TNF-α, IL-1Ra, IL-17A, and VEGF, but also several less well described cytokines/chemokines such as Fractalkine, Tie-2, and T cell chemokine CTACK. Importantly, we show here that 7 CCGs are significantly altered in SARS-CoV-2 exposed cancer patients (n = 52). Of these, TNF-α, IFN-β, TSLP, and sVCAM-1, identified to be elevated in haematological cancers, are also known tumour-promoting factors. Longitudinal analysis conducted over 3 months showed persistence of several tumour-promoting CCGs in SARS-CoV-2 exposed cancer patients. These data demonstrate a need for increased vigilance for haematological malignancy patients as a part of long COVID follow-up.

Blood cytokine analysis suggests that SARS-CoV-2 infection results in a sustained tumour promoting environment in cancer patients

Cytokines, chemokines and (angiogenic) growth factors (CCGs) have been shown to play an intricate role in the progression of both solid and haematological malignancies. Recent studies have shown that SARS-CoV-2 infection leads to worse outcome in cancer patients, especially in haematological malignancy patients. Here, we investigated how SARS-CoV-2 infection impacts the already altered CCG levels in solid or haematological malignancies, specifically whether there is a protective effect or rather a potentially higher risk for major COVID-19 complications in cancer patients due to elevated CCGs linked to cancer progression. Serially analysing immune responses with 55 CCGs in cancer patients under active treatment with or without SARS-CoV-2 infection, we first showed that cancer patients without SARS-CoV-2 infection (n=54) demonstrate elevated levels of 35 CCGs compared to the non-cancer, non-infected control group of health care workers (n=42). Of the 35 CCGs, 19 were common to both solid and haematological malignancy groups and comprised previously described cytokines such as IL-6, TNF-α, IL-1Ra, IL-17A, and VEGF, but also several less well described cytokines/chemokines such as Fractalkine, Tie-2, and T cell chemokine CTACK. Importantly, we show here that 7 CCGs are significantly altered in SARS-CoV-2 exposed cancer patients (n=52). Of these TNF-α, IFN-β, TSLP and sVCAM-1, identified to be elevated in haematological cancers, are also known tumour-promoting factors. Longitudinal analysis conducted over 3 months showed persistence of several tumour-promoting CCGs in SARS-CoV-2 exposed cancer patients. These data urge for increased vigilance for haematological malignancy patients as a part of long COVID follow-up.

A Partially Protective Vaccine for Fasciola hepatica Induced Degeneration of Adult Flukes Associated to a Severe Granulomatous Reaction in Sheep

Fasciolosis is an important economic disease of livestock. There is a global interest in the development of protective vaccines since current anthelmintic therapy is no longer sustainable. A better knowledge of the host-parasite interaction is needed for the design of effective vaccines. The present study evaluates the microscopical hepatic lesions in sheep immunized with a partially protective vaccine (VAC1), a non-protective vaccine (VAC2), and an infected control group (IC). The nature of granulomatous inflammation associated with degeneration of adult flukes found in the VAC1 group was characterized by immunohistochemistry. Hepatic lesions (fibrous perihepatitis, chronic tracts, bile duct hyperplasia, infiltration of eosinophils and lymphocytes and plasma cells) were significantly less severe in the VAC1 group than in the IC group. Dead adult flukes within bile ducts were observed only in the VAC1 group and were surrounded by a severe granulomatous inflammation composed by macrophages and multinucleate giant cells with a high expression of lysozyme, CD163 and S100 markers, and a low expression of CD68. Numerous CD3+ T lymphocytes and scarce infiltrate of FoxP3+ Treg and CD208+ dendritic cells were present. This is the first report describing degenerated flukes associated to a severe granulomatous inflammation in bile ducts in a F. hepatica vaccine trial.

An Enzyme-Linked Immunosorbent Assay for the Detection of Antibodies to Linguatula serrata in Experimentally Infected Dogs

Background: Linguatula serrata is a causative agent of visceral and nasopharyngeal linguatulosis in humans and animals. The aim of the present study was to investigate the immune response of dogs experimentally infected by L. serrata with ELISA. Methods: Five puppies were infected by inserting the L. serrata nymphs in their nasal cavities (infected group) in the Department of Parasitology of Shahid Chamran University of Ahvaz, during 2018-2019. Three animals were kept as the non-infected control group. Blood samples were collected from the animals for seven months at approximately monthly intervals for serum preparation. Nasal samples were taken weekly from the fourth month. ELISA was designed and performed on 64 sera (24 negatives, and 40 positives) using somatic (S), and excretory-secretory (ES) antigens. Results: Overall, 100% of the animals were infected with the parasite. Based on the results of ELISA, the ES antigen (sensitivity 95% and specificity 92%) was more preferred than the S antigen (sensitivity 95% and specificity 85%). Female parasites had significant effects on the immune response. There was a significant correlation between the clinical symptoms and the presence of female parasites (P<0.05). Conclusion: The results showed a practical method for dogs' experimental infection. ELISA method is suitable for the detection of infection at different stages of development, especially before the maturation stage of the parasite. In this regard, the ES antigen of the parasite was more immunogenic. Therefore, ELISA can be used as a serological method in the early detection and epidemiological studies of infection with L. serrata in dogs.

Prophylactic and Therapeutic Role of Vitamin D3 in Combination with Fluconazole Against Vaginal Candidiasis in a Murine Model

Objective: In the present study, we assessed the adjunct effect of vitamin D3 in the combination with fluconazole (FLZ) against vulvovaginal candidiasis (VVC) in mice. Methods: Prophylactic effect was assessed by pretreating mice with vitamin D3 before exposure of mice with 2 X 106 CFUs of Candida albicans followed by treatment with FLZ. To determine the combined therapeutic efficacy, C. albicans infected mice were treated with a combination of vitamin D3 (10 µg/kg) and FLZ (10 and 20 mg/kg). The efficacy of the treatment was assessed by analyzing the fungal load and blood cell count. Moreover, the levels of inflammatory cytokines, including IL-1β, IL-17 and TNF-α were analyzed in the vaginal tissues. The histological analysis of the vaginal tissue from the untreated and treated mice was performed to assess the efficacy of the treatment. Results: Prophylactic treatment with vitamin D3 (10 and 20 µg/kg) significantly increased the therapeutic effect of FLZ against VVC. In therapeutic experiment, mice in the infected control group showed the highest vaginal fungal load of 83627 ± 10058 CFUs. Treatment with FLZ at a dose of 10 mg/kg reduced fungal load to 55523 ± 14823 CFUs, whereas the mice treated with a combination of vitamin D3 and FLZ (10 mg/kg) had the fungal burden of 12156 ± 3219. Similarly, treatment with FLZ (20 mg/kg) reduced fungal load to 36394 ± 5648 CFUs, whereas the addition of vitamin D3 to FLZ (20 mg/kg) further reduced the fungal burden to 2179 ± 1188. The leukocyte numbers in the infected mice increased to 9802 ± 505 as compared to 5152 ± 778 in normal control mice. Whereas, a combination of vitamin D3 with FLZ (10 and 20 mg/kg) reduced leukocyte numbers to 7284 ± 607 and 5739 ± 1126. The histological analysis data revealed epithelial necrosis, shedding and ulceration in the vaginal wall. Treatment with FLZ or a combination of FLZ and vitamin D3 brought regenerative changes in the vaginal epithelium and lamina proparia. Conclusion: The results of the present work recommend that the addition of vitamin D3 may be considered to increase the efficacy of FLZ against VVC.

Efficacy of a multivalent vaccine against Fasciola hepatica infection in sheep

In this work we report the protection found in a vaccination trial performed in sheep with two different vaccines composed each one by a cocktail of antigens (rCL1, rPrx, rHDM and rLAP) formulated in two different adjuvants (Montanide ISA 61 VG (G1) and Alhydrogel®(G2)). The parameters of protection tested were fluke burden, faecal egg count and evaluation of hepatic lesions. In vaccinated group 1 we found a significant decrease in fluke burden in comparison to both unimmunised and infected control group (37.2%; p = 0.002) and to vaccinated group 2 (Alhydrogel®) (27.08%; p = 0.016). The lower fluke burden found in G1 was accompanied by a decrease in egg output of 28.71% in comparison with the infected control group. Additionally, gross hepatic lesions found in vaccine 1 group showed a significant decrease (p = 0.03) in comparison with unimmunised-infected group. The serological study showed the highest level for both IgG1 and IgG2 in animals from group 1. All these data support the hypothesis of protection found in vaccine 1 group.

Diclazuril-induced expression of CDK-related kinase 2 in the second-generation merozoites of Eimeria tenella

Background: Diclazuril is a classic anticoccidial drug. The key molecules of diclazuril in anticoccidial action allows target screening for the development of anticoccidial drugs. In the present study, a diclazuril anticoccidiosis animal model was established, and the transcription and translation levels of the CDK-related kinase 2 of Eimeria tenella (EtCRK2) were detected through quantitative real-time PCR and Western blot analysis, respectively. The localisation of EtCRK2 in merozoites was examined with immunofluorescence techniques.Results: The mRNA and protein expression levels of EtCRK2 decreased in the infected/diclazuril group compared with those in the infected/control group. In addition, immunofluorescence analysis showed that EtCRK2 was localised in the cytoplasm of the merozoites. The fluorescence intensity of EtCRK2 in the infected/diclazuril group was significantly weaker than that in the infected/control group.Conclusions: The anticoccidial drug diclazuril against E.tenella affects the expression pattern of EtCRK2 molecule, and EtCRK2 is a potential target for new drug development.

In vivo experimental infection of sarcosporidiosis and toxoplasmosis of rabbits in Duhok Province, Kurdistan region, Iraq

Background: Sarcocystis species and Toxoplasma gondii are both zoonotic obligatory intracellular protozoan organisms and cyst-forming coccidian parasites that occur in domestic animals and human throughout the world.Methods: Forty local breed rabbits were divided into four groups, each group ten. Group one were infected with Sarcocystis, group two with Toxoplasma and group three with both parasites and last group was non-infected control group. The LAT serological test was used for detection of anti-toxoplasma antibody in serum of Toxoplasma infected rabbits. The direct impression smears stained with Giemsa was prepared from different body organs including; liver, lung, heart, brain and skeletal muscle for detection of tissue cysts (Bradyzoites) of T. gondii and microcysts of Sarcocystis.Results: In group one, 70% of infected rabbits were positive for toxoplasmosis by serological test; both are and by impression smear method 80% of the rabbits were positive for T. gondii with tissue cysts. Fifty percent of rabbits were positive for microcysts of Sarcocystis by direct impression smear method in group two. In group three, the impression smear and latex agglutination method were positive in 40% and 60% of rabbits, respectively. Statistically, there was no significant difference in detection of toxoplasmosis and sarcocystosis by LAT and impression smear method in group one and three.Conclusions: Rabbits could be source of toxoplasmosis and sarcocystosis and have public health implications and hazard as source of food. They might be source of infection for cats and shed environmentally resistant oocysts.

Diclazuril-Induced Expression of CDK-Related Kinase 2 in the Second-Generation Merozoites of Eimeria Tenella

Background：Coccidiosis caused by Eimeria tenella infection, directly or indirectly leads to great loss to poultry industry. With the emergence of drug-resistance in chicken coccidia, it is imperative to develop new drugs. Cyclin-dependent kinases (CDKs) regulate cell cycle progression in numerous organisms by acting as key molecular switches. Results: In the present study, a diclazuril anticoccidiosis animal model was established and CDK-related kinase 2 (CRK2) in the second-generation merozoite of E. tenella (EtCRK2) gene was amplified through reverse transcription-polymerase chain reaction (RT-PCR) and expressed in Escherichia coli Rosetta (DE3). Purified recombinant protein was used for antiserum preparation. Subsequently, EtCRK2 transcription and translation levels were detected through quantitative real-time PCR and Western blot analysis, respectively. The localization of EtCRK2 in merozoites was examined via immunofluorescence techniques. Results showed that the mRNA and protein expression levels of EtCRK2 decreased in the infected/diclazuril group compared with those in the infected/control group. In addition, immunofluorescence analysis showed that EtCRK2 was localized in the cytoplasm of merozoites. The fluorescence intensity of EtCRK2 in the infected/diclazuril group was significantly weaker than that in the infected/control group. Conclusions: This study demonstrated that the anticoccidial drug diclazuril against E. tenella by affecting the expression pattern of EtCRK2 molecule, and EtCRK2 may be used as a candidate target for new drug development.

Efficacy of an Oral Solution Prepared from the Ultrasonic Extract of Radix dichroae roots against Eimeria tenella in Broiler Chickens

This study was conducted to determine the optimal dose of the oral solution of the ultrasonic extract of Radix dichroae (UERD) and to provide experimental support for a safe clinical dose for anticoccidial treatment of broiler chickens. Radix dichroae root extracts were prepared using the ultrasonic extraction method. The anticoccidial activity of the oral solution prepared from the ultrasonic extract of Radix dichroae roots was tested in broiler chickens following oral infection with a field isolate of E. tenella. Ninety Lingnan yellow broiler chickens (14 days old) were randomly divided into nine groups (n = 10), including six UERD oral solution treatments (0.25, 0.50, 1.50, 2.50, 3.50, and 5.00%), a toltrazuril group (0.10%), an E. tenella-infected control group, and a healthy control group. All groups were inoculated orally with 7 × 104 sporulated E. tenella oocysts (Guangdong strain) except for the healthy control group. The chickens in the seven drug-treated groups were administered a UERD oral solution or toltrazuril in drinking water for 7 days. The anticoccidial efficacy of the UERD oral solution was evaluated by the bloody diarrhoea severity level, relative body weight gain (rBWG), lesion score, oocyst per gram (OPG), and anticoccidial index (ACI). Compared with the infected control group, there were no significant differences in the groups treated with UERD oral solution or toltrazuril with regard to the lesion changes in the caecal regions (P>0.05); however, the blood contents, OPG, and oocyst score in three UERD oral solution treatment groups (0.50, 1.50, and 2.50%) were significantly reduced, and the bloody diarrhoea was also alleviated. The ACI in three UERD oral solution treatment groups (0.50%, ACI = 143.7; 1.50%, ACI = 151.0; and 2.50%, ACI = 144.3) was higher than that in the toltrazuril group (ACI = 127.0), and the rBWG in the 1.50% UERD oral solution treatment group (95.0%) was similar to that in the healthy control group (100%), which was also 12.5% higher than that in the toltrazuril group (82.5%). The findings of this study demonstrated that the UERD oral solution (0.50% ～ 2.50% dose range) showed better prevention, anticoccidial efficacy, and growth promotion effects than toltrazuril (0.10%), and the 1.50% dose level of UERD oral solution in water is the clinically recommended dose according to the present study conditions.