Evaluation of a Lateral Flow Assay for Rapid Detection of African Swine Fever Virus in Multiple Sample Types

Antibody-based lateral flow assay (LFA) is a quick and inexpensive tool used to detect pathogens in field samples, especially in hard-to-reach remote areas that may have limited access to central laboratories during an outbreak or surveillance. In this study, we investigated the ability of a commercially available LFA, PenCheck®, to detect African swine fever virus (ASFV) in clinical samples derived from pigs infected with highly virulent ASFV strains. The assay was specific and positively identified the majority of pigs showing high fever during the early stages (between 3 and 5 days) of infection. PenCheck® LFA also detected ASFV in serum and tissue samples collected from pigs that succumbed to experimental ASFV infection and whole blood, plasma, and tissue samples from the field. The limit of detection of the assay was ASFV titer 107.80 TCID50/mL, corresponding to ASFV real-time PCR values below 23 Ct. Although the sensitivity of the assay is less than that of the laboratory-based real-time PCR assays, the results obtained with the PenCheck® LFA in this study suggest that it can be used as a herd-level, field-deployable, and easy-to-use diagnostic tool to identify ASF-affected farms when access to portable molecular assays or central laboratories is not possible

Meat Exudate for Detection of African Swine Fever Virus Genomic Material and Anti-ASFV Antibodies

African swine fever (ASF) is one of the most important viral diseases of pigs caused by the ASF virus (ASFV). The virus is highly stable over a wide range of temperatures and pH and can survive in meat and meat products for several months, leading to long-distance transmission of ASF. Whole blood, serum, and organs from infected pigs are used routinely as approved sample types in the laboratory diagnosis of ASF. However, these sample types may not always be available. Here, we investigated meat exudate as an alternative sample type for the detection of ASFV-specific nucleic acids and antibodies. Pigs were infected with various ASFV strains: the highly virulent ASFV Malawi LIL 18/2 strain, the moderately-virulent ASFV Estonia 2014 strain, or the low-virulent ASFV OURT/88/3 strain. The animals were euthanized on different days post-infection (dpi), and meat exudates were collected and tested for the presence of ASFV-specific nucleic acids and antibodies. Animals infected with the ASFV Malawi LIL 18/2 developed severe clinical signs and succumbed to the infection within seven dpi, while pigs infected with ASFV Estonia 2014 also developed clinical signs but survived longer, with a few animals seroconverting before succumbing to the ASFV infection or being euthanized as they reached humane endpoints. Pigs infected with ASFV OURT/88/3 developed transient fever and seroconverted without mortality. ASFV genomic material was detected in meat exudate from pigs infected with ASFV Malawi LIL 18/2 and ASFV Estonia 2014 at the onset of viremia but at a lower amount when compared to the corresponding whole blood samples. Low levels of ASFV genomic material were detected in the whole blood of ASFV OURT/88/3-infected pigs, and no ASFV genomic material was detected in the meat exudate of these animals. Anti-ASFV antibodies were detected in the serum and meat exudate derived from ASFV OURT/88/3-infected pigs and in some of the samples derived from the ASFV Estonia 2014-infected pigs. These results indicate that ASFV genomic material and anti-ASFV antibodies can be detected in meat exudate, indicating that this sample can be used as an alternative sample type for ASF surveillance when routine sample types are unavailable or are not easily accessible

Thymic Stromal Lymphopoietin Is Critical for Regulation of Proinflammatory Cytokine Response and Resistance to Experimental Trypanosoma congolense Infection

African trypanosomiasis (sleeping sickness) poses serious threat to human and animal health in sub-Saharan Africa. Because there is currently no vaccine for preventing this disease and available drugs are not safe, understanding the mechanisms that regulate resistance and/or susceptibility to the disease could reveal novel targets for effective disease therapy and prevention. Thymic stromal lymphopoietin (TSLP) plays a critical role in driving Th2 immune response. Although susceptibility to experimental Trypanosoma congolense infection in mice is associated with excessive proinflammatory responses due in part to impaired Th2 response, the role of TSLP in resistance to African trypanosomiasis has not been well studied. Here, we investigated whether TSLP is critical for maintaining Th2 environment necessary for survival of T. congolense-infected mice. We observed an increased TSLP level in mice after infection with T. congolense, suggesting a role for this cytokine in resistance to the infection. Indeed, TSLPR−/− mice were more susceptible to T. congolense infection and died significantly earlier than their wild-type (WT) controls. Interestingly, serum levels of IFN-γ and TNF-α and the frequency of IFN-γ- and TNF-α-producing CD4+ T cells in the spleens and liver were significantly higher in infected TSLPR−/− mice than in the WT control mice. Susceptibility was also associated with excessive M1 macrophage activation. Treatment of TSLPR−/− mice with anti-IFN-γ mAb during infection abolished their enhanced susceptibility to T. congolense infection. Collectively, our study shows that TSLP plays a critical role in resistance to T. congolense infection by dampening the production of proinflammatory cytokines and its associated M1 macrophage activation

Regulatory T cells enhance susceptibility to experimental Trypanosoma congolense infection independent of mouse genetic background.

BACKGROUND: BALB/c mice are highly susceptible while C57BL/6 are relatively resistant to experimental Trypanosoma congolense infection. Although regulatory T cells (Tregs) have been shown to regulate the pathogenesis of experimental T. congolense infection, their exact role remains controversial. We wished to determine whether Tregs contribute to distinct phenotypic outcomes in BALB/c and C57BL/6 mice and if so how they operate with respect to control of parasitemia and production of disease-exacerbating proinflammatory cytokines. METHODOLOGY/FINDINGS: BALB/c and C57BL/6 mice were infected intraperitoneally (i.p) with 10(3)T. congolense clone TC13 and both the kinetics of Tregs expansion and intracellular cytokine profiles in the spleens and livers were monitored directly ex vivo by flow cytometry. In some experiments, mice were injected with anti-CD25 mAb prior or post T. congolense infection or adoptively (by intravenous route) given highly enriched naïve CD25(+) T lymphocytes prior to T. congolense infection and the inflammatory cytokine/chemokine levels and survival were monitored. In contrast to a transient and non significant increase in the percentages and absolute numbers of CD4(+)CD25(+)Foxp3(+) T cells (Tregs) in C57BL/6 mouse spleens and livers, a significant increase in the percentage and absolute numbers of Tregs was observed in spleens of infected BALB/c mice. Ablation or increasing the number of CD25(+) cells in the relatively resistant C57BL/6 mice by anti-CD25 mAb treatment or by adoptive transfer of CD25(+) T cells, respectively, ameliorates or exacerbates parasitemia and production of proinflammatory cytokines. CONCLUSION: Collectively, our results show that regulatory T cells contribute to susceptibility in experimental murine trypanosomiasis in both the highly susceptible BALB/c and relatively resistant C57BL/6 mice

Superficial Inguinal Lymph Nodes for Screening Dead Pigs for African Swine Fever

African swine fever (ASF) has spread across the globe and has reached closer to North America since being reported in the Dominican Republic and Haiti. As a result, surveillance measures have been heightened and the utility of alternative samples for herd-level monitoring and dead pig sampling have been investigated. Passive surveillance based on the investigation of dead pigs, both domestic and wild, plays a pivotal role in the early detection of an ASF incursion. The World Organization for Animal Health (OIE)-recommended samples for dead pigs are spleen, lymph nodes, bone marrow, lung, tonsil and kidney. However, obtaining these samples requires opening up the carcasses, which is time-consuming, requires skilled labour and often leads to contamination of the premises. As a result, we investigated the suitability of superficial inguinal lymph nodes (SILNs) for surveillance of dead animals. SILNs can be collected in minutes with no to minimum environmental contamination. Here, we demonstrate that the ASF virus (ASFV) genome copy numbers in SILNs highly correlate with those in the spleen and, by sampling SILN, we can detect all pigs that succumb to highly virulent and moderately virulent ASFV strains (100% sensitivity). ASFV was isolated from all positive SILN samples. Thus, sampling SILNs could be useful for routine surveillance of dead pigs on commercial and backyard farms, holding pens and dead on arrival at slaughter houses, as well as during massive die-offs of pigs due to unknown causes

Evaluation of a Lateral Flow Assay for Rapid Detection of African Swine Fever Virus in Multiple Sample Types

Antibody-based lateral flow assay (LFA) is a quick and inexpensive tool used to detect pathogens in field samples, especially in hard-to-reach remote areas that may have limited access to central laboratories during an outbreak or surveillance. In this study, we investigated the ability of a commercially available LFA, PenCheck®, to detect African swine fever virus (ASFV) in clinical samples derived from pigs infected with highly virulent ASFV strains. The assay was specific and positively identified the majority of pigs showing high fever during the early stages (between 3 and 5 days) of infection. PenCheck® LFA also detected ASFV in serum and tissue samples collected from pigs that succumbed to experimental ASFV infection and whole blood, plasma, and tissue samples from the field. The limit of detection of the assay was ASFV titer 107.80 TCID50/mL, corresponding to ASFV real-time PCR values below 23 Ct. Although the sensitivity of the assay is less than that of the laboratory-based real-time PCR assays, the results obtained with the PenCheck® LFA in this study suggest that it can be used as a herd-level, field-deployable, and easy-to-use diagnostic tool to identify ASF-affected farms when access to portable molecular assays or central laboratories is not possible

High and low dose infections preferentially expand CD4⁺ and CD8⁺ T cells, respectively.

<p>C57BL/6 mice were infected with low (1×10³) or high (2×10⁶) dose <i>L. major</i> promastigotes in their right hind footpad, sacrificed after 7 days and the draining lymph-node cells were labelled with CFSE dye and co-cultured with <i>L. major</i>-infected bone marrow-derived dendritic cells (BMDC) at a DC: lymph node cell ratio of 1∶100. After 4 days, the percentages of proliferating (CFSE^lo) CD4⁺ and CD8⁺ T cells (A and B) were assessed by flow cytometry. (A) is a representative histogram plots of individual animals while B represents the mean +/− SE of proliferating cells of all the animals (6 mice) within the group. Proliferating (CFSE^lo) IFN-γ (upper panels) and TNF (lower panels) -producing CD4⁺ (C and D) and CD8⁺ (E and F) T cells were also determined by flow cytometry. C and E are representative dot plots of individual animals within the group while D and F represent the mean +/− SE of proliferating cytokine-producing cells of all the animals (6 mice) within the group. The lymph nodes draining the infected feet were collected and digested with collagenase and the cells were then stained with different fluorochrome-conjugated antibodies against different DC subsets and the absolute numbers (upper panel) and percentage (lower panel) of CD11c⁺MHCII⁺ (G) and CD11c⁺CD103⁺CD8α⁺ (H) dendritic cells were determined by flow cytometry. Live cells were first gated on CD11c⁺ cells and further analyzed for MHC class II, CD103 and CD8α expression. Results are representative of 2 independent experiments (n = 6 mice/group) with similar results. *, p<0.05; **, p<0.01; ***, p<0.001.</p

CD8⁺ T cells Are Preferentially Activated during Primary Low Dose <i>Leishmania major</i> Infection but Are Completely Dispensable during Secondary Anti-<i>Leishmania</i> Immunity

<div><p>We previously showed that CD8⁺ T cells are required for optimal primary immunity to low dose <i>Leishmania major</i> infection. However, it is not known whether immunity induced by low dose infection is durable and whether CD8⁺ T cells contribute to secondary immunity following recovery from low dose infection. Here, we compared primary and secondary immunity to low and high dose <i>L. major</i> infections and assessed the influence of infectious dose on the quality and magnitude of secondary anti-<i>Leishmania</i> immunity. In addition, we investigated the contribution of CD8⁺ T cells in secondary anti-<i>Leishmania</i> immunity following recovery from low and high dose infections. We found that the early immune response to low and high dose infections were strikingly different: while low dose infection preferentially induced proliferation and effector cytokine production by CD8⁺ T cells, high dose infection predominantly induced proliferation and cytokine production by CD4⁺ T cells. This differential activation of CD4⁺ and CD8⁺ T cells by high and low dose infections respectively, was imprinted during <i>in vitro</i> and <i>in vivo</i> recall responses in healed mice. Both low and high dose-infected mice displayed strong infection-induced immunity and were protected against secondary <i>L. major</i> challenge. While depletion of CD4⁺ cells in mice that healed low and high dose infections abolished resistance to secondary challenge, depletion of CD8⁺ cells had no effect. Collectively, our results show that although CD8⁺ T cells are preferentially activated and may contribute to optimal primary anti-<i>Leishmania</i> immunity following low dose infection, they are completely dispensable during secondary immunity.</p></div