Triclabendazole efficacy, prevalence, and re-infection of Fasciola hepatica in bovine and ovine naturally infected in the Andes of Ecuador.

Fasciola spp., infections are distributed worldwide including the Andes region of Ecuador, affecting cattle, sheep, porcine, humans, and other herbivores. Triclabendazole (TCBZ) is commonly used to treat animal infections. However, prospective studies on TCBZ efficacy and fascioliosis prevalence have not been studied in the highlands of Ecuador. This study was performed in a rural community at central of the Ecuadorian Andes in freely roaming bovine and ovine aimed to 1) evaluate the efficacy of TCBZ by administering a single oral dose of 12 mg/kg body weight, 2) assess the prevalence of F. hepatica infection and 3) to monitor re-infections for a follow-up period of five months. In total, 122, 86, 111, 110, 89, and 90 and 49, 34, 47, 28, 27, and 31 stool samples were collected each month from bovines and ovine, respectively. Besides, 32 stool samples from porcine were also collected at the beginning of the study. Stools were microscopically analyzed by formalin-ether concentration method to detect F. hepatica ova. The prevalence of F. hepatica infections before treatment was 55,7% and 63,3% for bovine and ovine, respectively. The infection prevalence was of 22% in porcine. The efficacity of triclabendazole was 83% and 97% in bovines and ovine, respectively, at 30 days post-treatment. The re-infection reaches to 54,4% in bovines and 61,3% in ovine after five months. TCBZ had a high efficacy and could be used for bovines and ovine Fasciola infections in the study region; however, re-infections reach the initial prevalence after five months. Therefore, we recommend integrated control strategies, including chemotherapy with a single oral dose of TCBZ, vector control, and future drug resistance studies.

Survival persistence of the 3 common Salmonella enterica serotypes isolated from broilers' in different matrices.

Broiler meat is the predominant source of Salmonella as a foodborne pathogen. Several control strategies have focused on the reduction of Salmonella spp. levels at different production stages. However, the persistence of Salmonella between consecutive flocks is still of great concern. This study was designed to understand the cause of reinfection in broiler flocks due to survival of Salmonella in feeding lines of related matrices. Salmonella (S.) Enteritidis, S. Infantis, and S. Typhimurium isolated from broiler farms in North-West Germany were used. Four types of matrices (phosphate buffer saline (PBS), dietary plant fat, fat with feed mixture, and feed) were applied to evaluate Salmonella survival (with the initial dose about 8.0 log10 CFU/mL) during a simulation of 4 production cycles. To evaluate the growth and survival status of Salmonella ISO 6579-1:2017 were performed (quantitatively by plate count method (PCM) and most probable number method (MPN)) and qualitatively) at 5 defined time points (-7, 0, 4, 7, and 35 d). In all matrices and for the 3 serovars, the Salmonella count decreased at the end of the fourth cycle in comparison to the beginning of the experimental infection, and was still cultivated except for fat matrix. The PBS matrices showed the highest survival level of Salmonella and did not decline drastically by the end of the fourth cycle (5.93 ± 0.00, 5.87 ± 0.02, 5.73 ± 0.05 log10 CFU/mL, respectively). However, the fat matrices showed the lowest survival level for the 3 isolates at d 35 since the first cycle (0 log10 CFU/mL using PCM). Regarding the fat-feed mixture, and feed matrices, there was a fluctuation in the survival rate of Salmonella (all serovars) within each cycle. For the qualitative method, the 3 serovars persisted in all matrices until the end of the fourth cycle except for fat matrices. The present study highlights the ability of Salmonella to survive for a long time in different temperatures and matrices despite efficient cleaning and disinfection processes in the feeding lines, which may influence reinfection with Salmonella in poultry houses.

The dynamics of neutralizing antibodies against SARS-CoV-2 in cats naturally exposed to virus reveals an increase in antibody activity after re-infection.

Severe Acute Respiratory Syndrome Coronavirus 2 is the causative agent of Coronavirus Disease 2019 in humans. To date, little is known about the persistence of antibodies against SARS-CoV-2 in animals under natural conditions, in particular susceptible pets such as cat. This study reports the detection and monitoring of the humoral response against SARS-CoV-2 including the detection of immunoglobulins G specific for receptor binding domain of SARS-CoV-2 spike protein by an enzyme-linked immunosorbent assay and neutralizing antibodies by virus neutralization assay. Results showed that these antibodies last longer than 16 months in two naturally apparently healthy infected cats with the absence of clinicopathological findings during the follow-up. Moreover, re-infection is also possible with an important increase in virus neutralization test titers in both animals with no evident systemic signs found during each physical examination and with values of hematologic and biochemical parameters inside the normal reference intervals. Our results confirm a slow but progressive decrease of the kinetics and immunity of neutralizing antibodies in cats after the infection. Furthermore, similar to humans SARS-CoV-2 reinfection can stimulate an increase of the neutralizing antibodies determined by these two serological techniques in domestic cats.

Fasciola hepatica primoinfections and reinfections in sheep drive distinct Th1/Th2/Treg immune responses in liver and hepatic lymph node at early and late stages.

The expression of proinflammatory (IL-1ß, IFN-γ, TNF-α) and regulatory (IL-10, TGF-ß, IL-4) cytokines, as well as the transcription factor FoxP3, was quantified in the liver and hepatic lymph node (HLN) of sheep primoinfected and reinfected with Fasciola hepatica at early (4, 8 and 16 days post-infection [dpi]) and late (100 dpi) stages. The liver exerted a Th2 immune response at very early stages after the primoinfection with F. hepatica that induced the downregulation of IFN-γ, followed by a Th1/Th2/Treg response although the late stages were characterised by the expression of Th1/Th2 immune mediators. Contrarily, in reinfected sheep a robust mixed Th1/Th2/Treg immune response was found at very early stages meanwhile at late stages we observed a Th2/Treg immune response overcoming the expression of Th1 immune mediators. However, the HLN displayed a completely different Th1/Th2/Treg expression profile compared to the liver. Primoinfections with F. hepatica in HLN induced a mixed Th1/Th2/Treg environment from early stages, establishing a Th2 immune response at a late stage. However, the reinfected sheep exerted a Th2 immune response at early stages led by the IL-4 expression in opposition to the Th1/Th2/Treg found in the liver, meanwhile at late stages the HLN of reinfected sheep exerted a mixed Th1/Th2/Treg immune response. This is the first work publishing the expression of immune mediators in the liver and HLN from reinfected sheep with F. hepatica. The study of the immune responses exerted by the natural host in the target organs directly implied in the development of F. hepatica are crucial to better understand the immunopathogenesis of the fasciolosis being a key factor to develop effective vaccines.

Limited accrual of myxospores of Kudoa inornata (Cnidaria: Myxosporea) in their wild fish hosts, Cynoscion nebulosus (Teleostei: Sciaenidae).

Kudoa inornata is a myxosporean that infects the seatrout Cynoscion nebulosus. Increased prevalence of infection as fish age and absence of inflammation against plasmodia led to the hypothesis that seatrout retain and accumulate myxospores throughout their lives. However, opportunistic observations that wild-caught seatrout cleared infection when maintained in aquaculture conditions and evidence of encapsulated infected necrotic myofibers suggested that fish develop an immunity against this parasite, or that myxospores have a limited life span. To evaluate myxospore clearance and to test putative resistance to re-infection, we examined 44 wild-caught seatrout broodstock maintained in parasite-free water for 2-6 yr. Twenty-five fish served as negative controls (time zero of experiment), and 19 were exposed to water-borne K. inornata infective stages for 18 wk. Over 73% of the exposed fish became infected, compared to ~12% of control fish, indicating that fish were susceptible to re-infection by K. inornata. Whether plasmodia degenerate because K. inornata myxospores have a limited life span or seatrout develop an adaptive immunity against these life stages remains unknown. To test for accumulation of myxospores over time, we compared myxospore densities and intensities between sexes and across ages and sizes of wild seatrout. There was no significant difference in myxospore densities with size, age, or sex. However, intensities increased significantly with increasing fish age and size, indicating accrual of myxospores over time. These results combined with evidence of infection clearance suggest that K. inornata myxospores do not persist but nevertheless accrue in wild seatrout due to continuous contact with infective stages.

Porcine reproductive and respiratory syndrome virus reinfection causes the distribution of porcine interleukin-4 in close proximity to B lymphocytes within lymphoid follicles and a reduction in B and T lymphocytes.

Interleukin 4 (IL-4) plays a major role in T-lymphocyte development and is thought to be a central regulator as a cofactor in resting B-lymphocyte proliferation. Primary infection with porcine reproductive and respiratory syndrome virus (PRRSV) induces minimal IL-4 production, whereas an IL-4 response occurs in the peripheral blood of piglets reinfected by PRRSV. The locations and interaction partners for the massive volume of IL-4 triggered by PRRSV reinfection remain unclear. This study aimed to investigate the characteristics of IL-4 secretion and location changes in peripheral immune organs induced by PRRSV infection and reinfection. Our results show that PRRSV reinfection induced higher levels of IL-4 mRNA and protein expression in the peripheral immune organs (e.g., lymph node and spleen) and peripheral blood compared with PRRSV primary infection. Importantly, we found that, following PRRSV reinfection, an obvious large-scale migration of IL-4 occurred in the lymph nodes. During PRRSV primary infection, IL-4 was mainly concentrated around the lymphoid follicles and paracortical regions of the lymph node and also located in the marginal area and periarterial lymphatic sheath region of the spleen. During PRRSV reinfection, the now abundant IL-4 gathered into the lymphoid follicles of the lymph node and spleen. Notably, IL-4 changed its location state from scattered and sparse during primary infection to clinging to B lymphocytes in the lymphoid follicles during reinfection. During reinfection, IL-4 was often co-localized with T and B lymphocytes; furthermore, the percentages of several T lymphocyte subsets, N protein-specific antibody levels, and viral load in the peripheral blood or lymph tissues underwent remarkable variation. Another important finding of this study was that the numbers of B lymphocytes and T lymphocytes in the lymphoid nodes were significantly reduced after PRRSV infection or reinfection, presumably due to PRRSV-induced acute bone marrow failure and autophagy in thymic epithelial cells. This study revealed the characteristics of IL-4 migration and distribution in the peripheral lymph organs induced by PRRSV reinfection and provides valuable clues for further exploration of the interactions between IL-4, B lymphocytes, and T lymphocytes during PRRSV infection and reinfection.

SARS-CoV-2 superinfection and reinfection with three different strains.

We report a corona virus disease (COVID-19) case with unprecedented viral complexity. In the first severe episode, two different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains (superinfection) were identified within a week. Three months after discharge, the patient was readmitted and was infected in a nosocomial outbreak with a different strain, suffering a second milder COVID-19 episode.

Immune response does not prevent homologous Porcine epidemic diarrhoea virus reinfection five months after the initial challenge.

The aim of the present study was to evaluate the duration of protective immunity against Porcine epidemic diarrhoea virus (PEDV). To do so, a two phases study was performed. In the first phase, 75 four-week-old pigs (group A) were orally inoculated (0 days post-inoculation; dpi) with a European PEDV G1b strain and 14 were kept as controls (group B). The second phase started five months later (154 dpi), when animals in group A were homologous challenged and animals in group B were challenged for first time. Clinical signs, viral shedding and immune responses were evaluated after each inoculation, including the determination of antibodies (ELISA and viral neutralization test, IgA and IgG ELISPOTs using peripheral blood mononuclear cells and lymph node cells) and the frequency of interferon-gamma (IFN-γ) secreting cells. During the first phase, loose stools/liquid faeces were observed in all group A animals. Faecal shedding of PEDV occurred mostly during the first 14 days but, in some animals, persisted until 42 dpi. All inoculated animals seroconverted for specific-PEDV IgG and IgA, and for neutralizing antibodies (NA). At 154 dpi, 77% of pigs were still positive for NA. After that, the homologous challenge resulted in a booster for IgG, IgA, NA, as well as specific-PEDV IgG, IgA and IFN-γ secreting cells. In spite of that, PEDV was detected in faeces of all pigs from group A, indicating that the immune response did not prevent reinfection, although the duration of the viral shedding and the total load of virus shed were significantly lower for previously challenged pigs (p < .05). Taken together, the results indicated that, potentially, maintenance of PEDV infection within an endemic farm may occur by transmission to and from previously infected animals and also indicates that sterilizing immunity is shorter than the productive life of pigs.

Recurrences of multidrug-resistant tuberculosis: Strains involved, within-host diversity, and fine-tuned allocation of reinfections.

Recurrent tuberculosis occurs due to exogenous reinfection or reactivation/persistence. We analysed 90 sequential MDR Mtb isolates obtained in Argentina from 27 patients with previously diagnosed MDR-TB that recurred in 2018 (1-10 years, 2-10 isolates per patient). Three long-term predominant strains were responsible for 63% of all MDR-TB recurrences. Most of the remaining patients were infected by strains different from each other. Reactivation/persistence of the same strain caused all but one recurrence, which was due to a reinfection with a predominant strain. One of the prevalent strains showed marked stability in the recurrences, while in another strain higher SNP-based diversity was observed. Comparisons of intra- versus inter-patient SNP distances identified two possible reinfections with closely related variants circulating in the community. Our results show a complex scenario of MDR-TB infections in settings with predominant MDR Mtb strains.

Infection and reinfection of Stomoxys calcitrans larvae (Diptera: Muscidae) by entomopathogenic nematodes in different times of exposure.

Stomoxys calcitrans is a hematophagous dipteran. Several agents are used in biological control, including entomopathogenic nematodes (EPNs). Bioassay I involved an evaluation of the effect of Heterorhabditis bacteriophora HP88 and Heterorhabditis baujardi LPP7 on S. calcitrans larvae in different periods of exposure. Groups of 10 larvae were placed in Petri dishes and 200 EPNs/larva were added, which were divided into groups according to the exposure times of 2, 4, 6, 12, 24 and 48 hours. The purpose of Bioassay II was to evaluate the efficacy of the EPNs in infecting S. calcitrans larvae when they were isolated from stable fly larvae in Bioassay I. Groups of 10 larvae were placed in Petri dishes and 200 EPNs/larva were added. In bioassay I, H. bacteriophora caused mortality rates of 51.7, 83.3 and 91.7% in 12, 24 and 48 hours, respectively, while H. baujardi caused mortality rates of 9.3 (12h), 35 (24h) and 35% (48h). In Bioassay II, H. bacteriophora and H. baujardi resulted in mortality rates of 35% and 25%, respectively. It was concluded that the longest exposure times presented the highest larval mortality and that EPNs isolated from S. calcitrans are not efficient in controlling the larvae fly.

Experimental re-infected cats do not transmit SARS-CoV-2.

SARS-CoV-2 is the causative agent of COVID-19 and responsible for the current global pandemic. We and others have previously demonstrated that cats are susceptible to SARS-CoV-2 infection and can efficiently transmit the virus to naïve cats. Here, we address whether cats previously exposed to SARS-CoV-2 can be re-infected with SARS-CoV-2. In two independent studies, SARS-CoV-2-infected cats were re-challenged with SARS-CoV-2 at 21 days post primary challenge (DPC) and necropsies performed at 4, 7 and 14 days post-secondary challenge (DP2C). Sentinels were co-mingled with the re-challenged cats at 1 DP2C. Clinical signs were recorded, and nasal, oropharyngeal, and rectal swabs, blood, and serum were collected and tissues examined for histologic lesions. Viral RNA was transiently shed via the nasal, oropharyngeal and rectal cavities of the re-challenged cats. Viral RNA was detected in various tissues of re-challenged cats euthanized at 4 DP2C, mainly in the upper respiratory tract and lymphoid tissues, but less frequently and at lower levels in the lower respiratory tract when compared to primary SARS-CoV-2 challenged cats at 4 DPC. Viral RNA and antigen detected in the respiratory tract of the primary SARS-CoV-2 infected cats at early DPCs were absent in the re-challenged cats. Naïve sentinels co-housed with the re-challenged cats did not shed virus or seroconvert. Together, our results indicate that cats previously infected with SARS-CoV-2 can be experimentally re-infected with SARS-CoV-2; however, the levels of virus shed was insufficient for transmission to co-housed naïve sentinels. We conclude that SARS-CoV-2 infection in cats induces immune responses that provide partial, non-sterilizing immune protection against re-infection.

Infection and reinfection of Stomoxys calcitrans larvae (Diptera: Muscidae) by entomopathogenic nematodes in different times of exposure / Infecção e reinfecção de larvas de Stomoxys calcitrans (Diptera: Muscidae) por nematoides entomopatogênicos em diferentes tempos de exposição

Abstract Stomoxys calcitrans is a hematophagous dipteran. Several agents are used in biological control, including entomopathogenic nematodes (EPNs). Bioassay I involved an evaluation of the effect of Heterorhabditis bacteriophora HP88 and Heterorhabditis baujardi LPP7 on S. calcitrans larvae in different periods of exposure. Groups of 10 larvae were placed in Petri dishes and 200 EPNs/larva were added, which were divided into groups according to the exposure times of 2, 4, 6, 12, 24 and 48 hours. The purpose of Bioassay II was to evaluate the efficacy of the EPNs in infecting S. calcitrans larvae when they were isolated from stable fly larvae in Bioassay I. Groups of 10 larvae were placed in Petri dishes and 200 EPNs/larva were added. In bioassay I, H. bacteriophora caused mortality rates of 51.7, 83.3 and 91.7% in 12, 24 and 48 hours, respectively, while H. baujardi caused mortality rates of 9.3 (12h), 35 (24h) and 35% (48h). In Bioassay II, H. bacteriophora and H. baujardi resulted in mortality rates of 35% and 25%, respectively. It was concluded that the longest exposure times presented the highest larval mortality and that EPNs isolated from S. calcitrans are not efficient in controlling the larvae fly.

Resumo Stomoxys calcitrans é um díptero hematófago. Vários agentes são usados no controle biológico, incluindo nematoides entomopatogênicos (NEPs). O bioensaio I objetivou avaliar o efeito de Heterorhabditis bacteriophora HP88 e Heterorhabditis baujardi LPP7 sobre larvas de S. calcitrans em diferentes períodos de exposição. Grupos de 10 larvas foram colocados em placas de Petri, adicionou-se 200 NEPs/larva, e foram divididos de acordo com tempos de exposição de 2, 4, 6, 12, 24 e 48 horas. O objetivo do Bioensaio II foi avaliar a eficácia dos NEPs na infecção de larvas de S. calcitrans, quando esses foram isolados de larvas da mosca oriundos do Bioensaio I. Os grupos de 10 larvas foram colocados em placas de Petri e 200 NEPs/larva foram adicionados. No bioensaio I, H. bacteriophora causou taxas de mortalidade de 51,7%, 83,3% e 91,7% em 12, 24 e 48 horas, respectivamente, enquanto H. baujardi causou taxas de mortalidade de 9,3% (12h), 35% (24h) e 35% (48h). No Bioensaio II, H. bacteriophora e H. baujardi resultaram em taxas de mortalidade de 35% e 25%, respectivamente. Concluiu-se que os maiores tempos de exposição apresentaram as maiores mortalidades larvais e que NEPs isolados de S. calcitrans não são eficientes no controle das larvas mosca.

Dairy cattle with bovine leukaemia virus RNA show significantly increased leukocyte counts.

Infection with bovine leukaemia virus (BLV), a retrovirus, causes dysfunction of the immune system and can have a marked economic impact on dairy industries due to decreased milk production and reduced lifespan in affected dairy cattle. The presence of proviral DNA has been the major diagnostic indicator of BLV infection. However in the course of BLV infection, the viral genome can be dormant, without detectable gene expression, resulting in limited impact on infected animals. At present, there is limited knowledge regarding haematological indices in dairy cattle that could indicate activation of the BLV genome and suggest reactivated BLV infection. In this study, BLV infection and BLV genome reactivation were evaluated based on the presence of BLV DNA and BLV env gene transcripts, respectively. BLV RNA transcription was confirmed. Among 93 whole blood samples obtained from asymptomatic dairy cattle, the prevalence of BLV proviral DNA and transcripts was 93.5% (n = 87/93) and 83.9% (n = 78/93), respectively. Between groups with and without BLV, the mean counts of white blood cells and lymphocytes in whole blood were significantly associated with the presence of BLV RNA (P < 0.05), but not with BLV proviral DNA. These results shed light on the activation status of the BLV genome and should be taken into account when evaluating the possible impact of BLV on cattle.