The resting sites and blood-meal sources of Anopheles minimus in Taiwan

<p>Abstract</p> <p>Background</p> <p>The WHO declared Taiwan free from malaria in 1965, but in 2003 the reporting of two introduced cases in a rural area suggested a possible local transmission of this disease. Therefore, understanding the resting sites and the blood sources of <it>Anopheles minimus </it>is crucial in order to provide information for implementing vector control strategies.</p> <p>Methods</p> <p>During a two-year survey, mosquitoes were collected in houses and their surrounding areas and at the bank of larval habitats by backpack aspirators in 17 villages in rural areas of southern and eastern Taiwan for 1 hr. On the same day, blacklight traps were hung downward overnight. Blood-fed mosquito samples were analysed by PCR.</p> <p>Results</p> <p>Of the 195 total households surveyed by backpack aspirators, no <it>Anopheles </it>adults were collected inside the houses, while a single <it>Anopheles minimus </it>and a single <it>Anopheles maculatus </it>were collected outside of the houses. On the same day, 23 <it>An. minimus</it>, two <it>An. maculatus</it>, two <it>Anopheles ludlowae</it>, two <it>Anopheles sinensis</it>, and one <it>Anopheles tessellatus </it>were collected along the bank of larval habitats. In blacklight traps hung outside of the houses in the villages, 69 <it>An. minimus</it>, 62 <it>An. ludlowae</it>, 31 <it>An. sinensis</it>, and 19 <it>An. maculatus </it>were collected. In larval habitats, 98 <it>An. ludlowae</it>, 64 <it>An. minimus</it>, 49 <it>An. sinensis</it>, and 14 <it>An. maculatus </it>were collected. Of a total of 10 blood-fed samples, <it>An. minimus </it>fed on four animals including bovine (60%), dogs (20%), pig (10%), and non-chicken avian (10%).</p> <p>Conclusion</p> <p><it>Anopheles minimus</it>, an opportunist feeder in Taiwan, was not collected inside the houses, but was found outside of the houses in villages and surrounding larval habitats. Therefore, an outdoor transmission of malaria is likely to occur and, thus, the bed nets, which are favoured for controlling the late biting of <it>An. minimus</it>, should be a very efficient and effective method for those local residents who sleep outdoors. Additionally, space spray of insecticides for <it>Anopheles </it>at night, as well as residual spray inside animal huts and selective larval habitats, are also helpful to control female adults.</p

Development and evaluation of a loop-mediated isothermal amplification method for rapid detection and differentiation of two genotypes of porcine circovirus type 2

BackgroundPorcine circovirus type 2 (PCV2) is one of the major swine viral diseases and has caused significant economic loss to pig producers. PCV2 has been divided into two major genotypes: PCV2a, PCV2b. A loop-mediated isothermal amplification (LAMP) method was developed for the detection and differentiation of PCV2a and PCV2b in clinical samples.MethodsLAMP-specific primer sets were designed based on six PCV2a and six PCV2b reference isolates. To determine the analytical specificity of LAMP, DNA samples extracted from 36 porcine virus isolates were tested by LAMP, including eight PCV2a, 11 PCV2b, four PCV type 1, two porcine parvovirus, three pseudorabies virus, and eight porcine reproductive and respiratory virus. To evaluate the analytical sensitivity of the assay, 10-fold serial dilutions of PCV2a and PCV2b recombinant plasmids were performed to prepare the dilutions at concentration from 106 to 1 copy(ies)/μL, and each dilution was tested by both LAMP and nested polymerase chain reaction (nested PCR). A total of 168 clinical samples were analyzed by both LAMP and nested PCR, and the relative sensitivity and specificity of LAMP compared to nested PCR were calculated.ResultsUsing different primer sets of LAMP, LAMP could be completed within 50 minutes. This method was found to be highly analytically specific for PCV2a and PCV2b; only the target gene was detected without cross-reaction. The analytical sensitivity of LAMP for PCV2a and PCV2b were 10 copies/μL, demonstrating analytical sensitivity comparable to that obtained using nested PCR. In addition, the sensitivity and specificity of LAMP relative to those of nested PCR were 97.7% and 100.0%, respectively. The percentage of observed agreement was 98.2%, and the κ statistic was 0.949.ConclusionLAMP is a rapid, specific, and sensitive diagnostic method for the detection and differentiation of PCV2a and PCV2b in clinical samples

Porcine circovirus type 2 (PCV2) infection decreases the efficacy of an attenuated classical swine fever virus (CSFV) vaccine

The Lapinized Philippines Coronel (LPC) vaccine, an attenuated strain of classical swine fever virus (CSFV), is an important tool for the prevention and control of CSFV infection and is widely and routinely used in most CSF endemic areas, including Taiwan. The aim of this study was to investigate whether PCV2 infection affects the efficacy of the LPC vaccine. Eighteen 6-week-old, cesarean-derived and colostrum-deprived (CDCD), crossbred pigs were randomly assigned to four groups. A total of 105.3 TCID50 of PCV2 was experimentally inoculated into pigs through both intranasal and intramuscular routes at 0 days post-inoculation (dpi) followed by LPC vaccination 12 days later. All the animals were challenged with wild-type CSFV (ALD stain) at 27 dpi and euthanized at 45 dpi. Following CSFV challenge, the LPC-vaccinated pigs pre-inoculated with PCV2 showed transient fever, viremia, and viral shedding in the saliva and feces. The number of IgM+, CD4+CD8-CD25+, CD4+CD8+CD25+, and CD4-CD8+CD25+ lymphocyte subsets and the level of neutralizing antibodies against CSFV were significantly higher in the animals with LPC vaccination alone than in the pigs with PCV2 inoculation/LPC vaccination. In addition, PCV2-derived inhibition of the CSFV-specific cell proliferative response of peripheral blood mononuclear cells (PBMCs) was demonstrated in an ex vivo experiment. These findings indicate that PCV2 infection decreases the efficacy of the LPC vaccine. This PCV2-derived interference may not only allow the invasion of wild-type CSFV in pig farms but also increases the difficulty of CSF prevention and control in CSF endemic areas

Dentinogenic ghost cell tumor in a Sumatran Rhinoceros

An adult female Sumatran rhinoceros was observed with a swelling in the left infraorbital region in March 2017. The swelling rapidly grew into a mass. A radiograph revealed a cystic radiolucent area in the left maxilla. In June 2017, the rhinoceros was euthanized. At necropsy, the infraorbital mass measured 21 cm × 30 cm. Samples of the infraorbital mass, left parotid gland, and left masseter muscle were collected for histopathology (Hematoxylin & Eosin, Von Kossa, Masson’s trichrome, cytokeratin AE1/AE3, EMA, p53, and S-100). Numerous neoplastic epithelial cells showing pleomorphism and infiltration were observed. Islands of dentinoid material containing ghost cells and keratin pearls were observed with the aid of the two special histochemistry stains. Mitotic figures were rarely observed. All the neoplastic odontogenic cells and keratin pearls showed an intense positive stain for cytokeratin AE1/AE3, while some keratin pearls showed mild positive stains for S-100. All samples were negative for p53 and S-100 immunodetection. The mass was diagnosed as a dentinogenic ghost cell tumor

Th2 cytokine bias induced by silver nanoparticles in peripheral blood mononuclear cells of common bottlenose dolphins (Tursiops truncatus)

Background Silver nanoparticles (AgNPs) have been widely used in many commercial products due to their excellent antibacterial ability. The AgNPs are released into the environment, gradually accumulate in the ocean, and may affect animals at high trophic levels, such as cetaceans and humans, via the food chain. Hence, the negative health impacts caused by AgNPs in cetaceans are of concern. Cytokines play a major role in the modulation of immune system and can be classified into two types: Th1 and Th2. Th1/Th2 balance can be evaluated by the ratios of their polarizing cytokines (i.e., interferon [IFN]-γ/Interleukin [IL]-4), and animals with imbalanced Th1/Th2 response may become more susceptible to certain kinds of infection. Therefore, the present study evaluated the in vitro cytokine responses of cetacean peripheral blood mononuclear cells (cPBMCs) to 20 nm citrate-AgNPs (C-AgNP20) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Methods Blood samples were collected from six captive common bottlenose dolphins (Tursiops truncatus). The cPBMCs were isolated and utilized for evaluating the in vitro cytokine responses. The cytokines evaluated included IL-2, IL-4, IL-10, IL-12, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. The geometric means of two housekeeping genes (HKGs), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β2-microglobulin (B2M), of each sample were determined and used to normalize the mRNA expression levels of target genes. Results The ratio of late apoptotic/necrotic cells of cPBMCs significantly increased with or without concanavalin A (ConA) stimulation after 24 h of 10 µg/ml C-AgNP20 treatment. At 4 h of culture, the mRNA expression level of IL-10 was significantly decreased with 1 µg/ml C-AgNP20 treatment. At 24 h of culture with 1 µg/ml C-AgNP20, the mRNA expression levels of all cytokines were significantly decreased, with the exceptions of IL-4 and IL-10. The IFN-γ/IL-4 ratio was significantly decreased at 24 h of culture with 1 µg/ml C-AgNP20 treatment, and the IL-12/IL-4 ratio was significantly decreased at 4 or 24 h of culture with 0.1 or 1 µg/ml C-AgNP20 treatment, respectively. Furthermore, the mRNA expression level of TNF-α was significantly decreased by 1 µg/ml C-AgNP20 after 24 h of culture. Discussion The present study demonstrated that the sublethal dose of C-AgNP20 (≤1 µg/ml) had an inhibitory effect on the cytokine mRNA expression levels of cPBMCs with the evidence of Th2 cytokine bias and significantly decreased the mRNA expression level of TNF-α. Th2 cytokine bias is associated with enhanced immunity against parasites but decreased immunity to intracellular microorganisms. TNF-α is a contributing factor for the inflammatory response against the infection of intracellular pathogens. In summary, our data indicate that C-AgNP20 suppresses the cellular immune response and thereby increases the susceptibility of cetaceans to infection by intracellular microorganisms

Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro

Granulomatous lymphadenitis is one of the pathognomonic lesions in post-weaning multisystemic wasting syndrome (PMWS)-affected pigs. This unique lesion has not been reported in direct association with viral infection in pigs. The objective of the present study was to evaluate whether porcine circovirus type 2 (PCV2) alone is able to induce functional modulation in porcine monocytic cells in vitro to elucidate its possible role in the development of granulomatous inflammation. It was found that the proliferation activity of blood monocytes (Mo) and monocyte-derived macrophages (MDM) was significantly enhanced by PCV2. During monocyte-macrophage differentiation, the PCV2 antigen-containing rate and formation of multinucleated giant cells (MGC) were significantly increased in MDM when compared to those in Mo. The MDM-derived MGC displayed a significantly higher PCV2 antigen-containing rate than did the mono-nucleated MDM. Supernatants from PCV2-inoculated MDM at 24 h post-inoculation induced an increased tendency of chemotactic activity for blood Mo. At the same inoculation time period, levels of mRNA expression of the monocytic chemokines, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1, also significantly increased in PCV2-inoculated MDM. The results suggest that PCV2 alone may induce cell proliferation, fusion, and chemokine expression in swine monocytic cells. Thus, PCV2 itself may play a significant role in the induction of granulomatous inflammation in PMWS-affected pigs

Detection of porcine circovirus type 1 in commercial porcine vaccines by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) method with a real-time monitoring system was developed for the detection of porcine circovirus type 1 (PCV1) in commercial swine vaccines. This method was highly specific for PCV1. No cross-reaction to porcine circovirus type 2, porcine parvovirus, pseudorabies virus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus was observed. The analytical sensitivity of the LAMP for PCV1 DNA was 10 copies/μl in the case of positive recombinant plasmid comparable to that obtained from the nested polymerase chain reaction (nested PCR). Furthermore, 25 commercial swine vaccines were tested by both the LAMP and the nested PCR, and three of them were tested positive for PCV1 DNA. These results indicate that PCV1 DNA can be real-time detected by the LAMP; the method was highly specific, sensitive, and rapid for the detection of PCV1 DNA, particularly in commercial swine vaccines

The effect of infection order of porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus on dually infected swine alveolar macrophages

Abstract Background: Concurrent infection with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) is known as one of the major causes for porcine respiratory disease complex (PRDC). Dual infection with PCV2 and PRRSV is consistently to have more severe clinical presentations and pulmonary lesions than infection with PCV2 alone or PRRSV alone. However, it is not known if dual infections with PCV2 and PRRSV in different infection order may lead to different clinical symptoms in the host. To mimic the possible field conditions, swine alveolar macrophages (AMs) were inoculated with PCV2 and PRRSV in vitro simultaneously or with one virus 18 h earlier than the other. The cell viability, cytopathic effects, antigen-containing rates, phagocytotic and microbial killing capabilities, cytokine profiles (IL-8, TNF-α, and IFN-α) and FasL transcripts were determined, analyzed, and compared to prove the hypothesis. Results: A marked reduction in PRRSV antigen-containing rate, cytopathic effect, and TNF-α expression level was revealed in AMs inoculated with PCV2 and PRRSV simultaneously and in AMs inoculated with PCV2 first then PRRSV 18 h later, but not in AMs inoculated with PRRSV first then PCV2 18 h later. Transient decrease in phagocytosis but constant reduction in microbicidal capability in AMs in the group inoculated with PCV2 alone and constant decrease in phagocytosis and microbicidal capability in AMs in all PRRSV-inoculated groups were noted. The levels of IL-8, TNF-α, IFN-α, and FasL transcripts in AMs in all groups with dual inoculation of PCV2 and PRRSV were significantly increased regardless of the infection orders as compared with infection by PCV2 alone or PRRSV alone. Conclusions: Swine AMs infected with PCV2 first then PRRSV later or infected with PCV2 and PRRSV simultaneously displayed marked reduction in PRRSV antigen-containing rate, cytopathic effect, and TNF-α expression level. The different inoculation orders of PCV2 and PRRSV in AMs leading to different results in viral antigen positivity, cytopathology, and cytokine profile may explain, at least partially, the underlying mechanism of the enhanced pulmonary lesions in PRDC exerted by dual infection with PCV2 and PRRSV and the variable clinical manifestations of PRDC-affected pigs in the field. Keywords: Porcine circovirus type 2, Porcine reproductive and respiratory syndrome virus, Alveolar macrophages, Porcine respiratory disease complex

Dentinogenic Ghost Cell Tumor in a Sumatran Rhinoceros

An adult female Sumatran rhinoceros was observed with a swelling in the left infraorbital region in March 2017. The swelling rapidly grew into a mass. A radiograph revealed a cystic radiolucent area in the left maxilla. In June 2017, the rhinoceros was euthanized. At necropsy, the infraorbital mass measured 21 cm × 30 cm. Samples of the infraorbital mass, left parotid gland, and left masseter muscle were collected for histopathology (Hematoxylin &amp; Eosin, Von Kossa, Masson’s trichrome, cytokeratin AE1/AE3, EMA, p53, and S-100). Numerous neoplastic epithelial cells showing pleomorphism and infiltration were observed. Islands of dentinoid material containing ghost cells and keratin pearls were observed with the aid of the two special histochemistry stains. Mitotic figures were rarely observed. All the neoplastic odontogenic cells and keratin pearls showed an intense positive stain for cytokeratin AE1/AE3, while some keratin pearls showed mild positive stains for S-100. All samples were negative for p53 and S-100 immunodetection. The mass was diagnosed as a dentinogenic ghost cell tumor