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No specific medical therapy has proven beneficial once people become ill from bat EIDs (at least of viral origin). For example, although rabies is an ancient disease, effective therapeutic treatment of rabies in humans continues to be very challenging. Rapid early diagnosis in the biting animal is critical, since identification of rabies before its fulminant stage allows for effective prophylaxis. Fulminant rabies continues to carry a very poor prognosis. The first case of the successful experimental treatment of rabies in a naïve patient was a 15-year-old girl bitten by a bat in 2004 (145). However, extension of the ‘Milwaukee Protocol’ (i.e., therapeutic coma, antiviral drugs, intensive medical care) in other patients has been much less successful (see for example Rupprecht (146) and Rubin et al. (147)).
Prophylaxis, after exposure but well in advance of illness, has a much higher success rate. Appropriate post-exposure wound cleansing has been shown to reduce significantly the likelihood of RABV transmission (148). Besides washing the wound with soap and water, unvaccinated persons should receive both rabies immune globulin and four doses of cell-culture vaccine. Globally, more than 12 million persons receive post-exposure prophylaxis each year (149).
Besides rabies, novel treatment strategies are being developed for other bat EIDs. The use of RNA interference has been suggested for the treatment of henipaviruses (150). These currently untreatable infections may be ameliorated by the introduction of small interfering RNA molecules homologous to the RNA in these pathogens. While promising in theory for many agents, this line of treatment is still in its preliminary stages, and issues such as efficacy in humans, delivery, and cost have yet to be addressed.
The potential for filoviruses to be used as bioweapons has spurred research efforts for an effective vaccine that could be used in an outbreak. For example, in a mouse model of hemorrhagic EBOV infection, a vesicular stomatitis virus-based vaccine has been shown to be safe and effective in preventing clinical presentation of disease (151). Furthermore, the possibility that this vaccine may be deliverable through mucosal surfaces offers potential as a rapid vaccination agent during an outbreak.
Several review articles have described and discussed animal models for MERS-CoV infection,,,. In this section, the current status of animal models for MERS disease reproduction is briefly summarized.
After the identification of MERS-CoV in 2012, the efforts were directed to develop an animal model to study pathogenesis and to test the efficacy of vaccines and/or treatments in vivo. Similar to SARS-CoV, rhesus macaques have demonstrated susceptibility to MERS-CoV,,. A work led by Munster demonstrated that the common marmoset is also suitable as a MERS-CoV model. They showed that this model recapitulates the disease observed in humans; therefore, findings in the evaluation of potential therapeutic strategies might be implemented in humans. However, small animals are required for controlled, large and comprehensive studies. While, at first, experiences with SARS-CoV turned out to be very helpful for the research on MERS-CoV, the development of a small animal model for MERS was a more difficult task,. Raj and collaborators rapidly identified dipeptidyl peptidase-4 (DPP4) as the functional receptor for MERS-CoV, and DPP4 is present in lung cells of many rodents. Thus, rodents were expected to be susceptible for MERS-CoV. However, and as predicted by the crystal structure analysis of the MERS-CoV receptor binding domain (RBD) with the human DPP4 (hDDP4) extracellular domain, so far, no rodent model is naturally permissive for MERS-CoV infection. In Syrian hamster, the DPP4 receptor was shown to be expressed on bronchiolar epithelium, but inoculation of MERS-CoV via aerosols or intratracheal routes with different doses did not lead to productive infection. Wild type and immune-deficient mice were also tested for MERS-CoV infection without success. Since then, several groups have been focused on new strategies to develop a small animal model susceptible to MERS-CoV infection. It was found that mouse cells could be made permissive for MERS-CoV when expressing hDPP4. Consequently, the hDPP4 was transduced into mouse lungs using an adenovirus vector, which resulted in animals susceptible to MERS-CoV infection. These mice exhibited pneumonia and extensive inflammatory-cell infiltration with the presence of virus in the lungs. Recently, a transgenic mice model expressing hDPP4, highly susceptible to MERS-CoV infection and able to display systemic lesions, has been developed. As demonstrated for several diseases, transgenic animal models have become an important tool to improve medical research. On the other hand, glycosylation of the murine DPP4 is a major factor impacting the receptor function by blocking the binding to MERS-CoV. Therefore, the modification of the mouse genome to match the sequence in the hDPP4 made this species susceptible to MERS-CoV infection. Accordingly, these newly established mice models are useful to evaluate the efficacy of vaccines and therapeutic agents against MERS-CoV infection,,,. VelocImmune and VelociGene technologies have been used to develop a humanized mouse model for MERS-CoV infection; these methodologies can be also applied for other pathogens in future emerging epidemics.
The pre-publication history for this paper can be accessed here:
A school-aged patient with a previous history of mild eczema developed a respiratory tract infection in October 2016, a couple of days after visiting a pig farm. The child had entered the pigsty but had not been in direct contact with pigs. Despite early prescription of antibiotics by the general practitioner the child’s clinical situation rapidly deteriorated. Within three days after onset of disease the child was transferred to a paediatric intensive care unit (PICU) for non-invasive ventilation support and intensive monitoring. Despite these efforts, the patient deteriorated further and was intubated in order to start mechanical ventilation. Bronchoscopy following intubation revealed large amounts of highly viscous mucus in the airways. Efforts to remove this mucus failed to improve ventilation. Mechanical ventilation became increasingly complex and it was decided to initiate veno-venous extracorporeal membrane oxygenation (ECMO) and to transfer to a quaternary PICU. Due to ECMO, blood oxygenation was secured and extensive bronchoscopy could be performed, during which topical DNAse (Dornase alpha, Pulmozyme, Roche) was instilled to decrease viscosity and facilitate removal of obstructing mucus plugs. On the following day, bronchoscopy was repeated and additional mucus was removed.
In the days following these procedures, the patient improved rapidly. ECMO was discontinued five days after start and the patient could be extubated. For the entire duration of hospitalisation, the patient had received broad-spectrum antibiotics, although all bacterial cultures remained negative. Throat swabs had been collected at initial admission and tested positive for influenza A virus, of which the quaternary PICU was informed on the day after the patient transfer. Oseltamivir treatment (60 mg twice daily) was started hours after initiation of ECMO and transport. It was continued for a total of 7 days when a nasal swab tested negative for influenza virus. At the time of submission of this report, the child was recovering well.
Chloroquine is a drug widely used in the past in the antimalarial therapy and prophylaxis before the emergence of resistant Plasmodium spp strains. This drug, readily available and well tolerated, is also endowed with antiviral properties, acting at two levels: the entry step and the inflammation process.
Indeed, chloroquine is a lysosomotropic agent that increases the endosomal pH affecting the normal vesicle sorting and endosome-membrane fusion. Furthermore, chloroquine displays anti-inflammatory properties by down-regulating the production of cytokines (IFN-γ and TNF-α), and the expression of TNF-α receptor. Thus, the antiviral activity of chloroquine could be effective towards all viruses that require an acidic pH for infection of host cells, such as EBOV, and mitigate the clinical signs due to the deleterious strong immune activation following viral infection. The anti-EBOV activity of chloroquine has been reported in several in vitro studies adopting different viral models and cellular targets (reviewed in). Despite promising evidence, in vivo studies did not fully support the efficacy of chloroquine for the treatment of EBOV infection. In fact, the encouraging results from two studies by Madrid and co-workers, showing a protective effect of chloroquine in mice infected with a mouse-adapted EBOV strain were not supported by more recent data, based on similar regimens, in mice, hamsters and the guinea pigs. As well as other CADs, chloroquine may be tested for prophylactic treatment considering that it should accumulate inside host cells to display the antiviral activity.
Among drugs correlated with chloroquine, amodiaquine, hydroxychloroquine, and aminoquinoline have been shown to inhibit filovirus infection in vitro using a pseudotyped virus assay and the authentic EBOV. Although no in vivo experiments have been undertaken yet, a promising result was obtained by a retrospective analysis performed on patients treated in Liberia with artesunate-amodiaquine during the Western Africa outbreak of EVD. In fact, these patients showed a lower risk of death from EVD than patients treated with artemether-lumefantrine. Although this observation lacks of several controls, the clinical effect of the artesunate-amodiaquine treatment should be better investigated as a possible therapeutic option for patients with EVD.
Recently, Lee and coworkers reported that the new antimalarial drug ferroquine inhibits EBOV entry, by affecting the pH dependent viral fusion step.
Suramin is a drug adopted to treat the trypanosome-caused African blindness. It has been demonstrated that Suramin, as a competitive inhibitor of heparin, displays antiviral activity and inhibits Chikungunya virus and EBOV infection in cellular models. However, due to its significant side effects, Suramin should be taken into consideration as therapeutic option only for highly deadly viral infections.
The FDA-approved compound Emetine, used for the treatment of amoebiasis, and its structural desmethyl analog have been shown to accumulate into the endosome/lysosome compartment inhibiting EBOV infection.
Finally, by using a VLP-based approach, the anthelmintic drugs albendazole and mebendazole have been reported to inhibit EBOV infection.
Inactivated vaccines are safer than live vaccines because they cannot replicate at all in a vaccinated host, resulting in no risk of reversion to a virulent form capable of causing diseases. However, they generally provide a shorter length of protection than live vaccine and generally elicit weak immune responses, in particular cell-mediated immunity, as opposed to live viral vaccines. For this reason, inactivated vaccines are administered with potent adjuvant, and require boosters to elicit satisfactory and a long-term immunity. Vaccines of this type are generally created by inactivating propagated viruses by treatment with heat or chemicals such as formalin or binary ethyleneimine. This procedure can destroy the pathogen's ability to propagate in the vaccinated host, but keeps it intact so that the immune system can still recognize it. Although inactivated virus vaccines have been used for preventing various types of viral diseases over the decades, they need further development for controlling newly emerging diseases.
For examples, influenza virus vaccines are continually improved to contain all serotypes because many new serotypes emerge in new outbreaks. As with other approaches, many studies have been focused on searching for better adjuvants which enhance immune responses in accordance with inactivated vaccines as well as help to overcome the inhibitory effects of maternal antibody. For live AIV vaccines, the possibility of reassortment between live vaccine strain and field isolates and of back mutation from low-pathogenic to highly pathogenic viruses lead to serious concerns for vaccine safety. Thus, prior stimulation of the immune system using some immunomodulators followed by vaccination with inactivated vaccines may be needed to confer better protective immunity within a short period of time and may be promising in controlling LPAI H9N2.
Vaccination has been proven to be a cost-effective means to prevent infectious diseases and eradicate such infectious agents. As stated in this article, use of vaccines in industry animals has greatly influenced animal health, welfare, productivity of industry animals, and eventually contributed to food safety. Despite excellent illustration of vaccine effectiveness against various pathogens, development of animal vaccines has been recognized to be a challenging task due to the presence of a variety of animal types, infectious agents, and different pathogenic mechanisms for each pathogen. In some cases, animal scientists contribute to the development of human vaccines by providing research results obtained from experimental animals, as well as by attempting new trials on animals (which is less restricted than human trials). Due to their similar size and anatomy to humans, large animals, in particular pigs are useful for testing vaccine efficacy and vaccine delivery systems. The ultimate goal of animal vaccines is to provide efficient protection to various animals from such infectious agents. For this reason, development of animal vaccines should be achieved by multidisciplinary collaboration, including microbiology, immunology, proteomics, genetics, molecular biology, and even bioinformatics.
Although it is expected to elicit immune responses to protect from wild type infection, vaccination may not be able to completely prevent a natural infection but might reduce the severity of the disease. Sometimes, vaccination can fail when it is executed with improper timing (such as high levels of pre-existing antibody, malnutrition, environment extremes, and stress conditions), resulting in adverse effects and poor immune responses. As another factor, vaccines that do not contain proper immunogenic antigens will not be effective, and therefore correct vaccines should be selected and applied for the proper of time. Adverse effects derived from vaccination should be minimal as well as acceptable. The cost of vaccination should be less than economic loss induced by naturally infection. Additionally, in order to effectively cope with the treatment of various infectious agents, vaccination should be used for animals, with the mutual exchange of information among practitioners, farmers, and disease control agencies. In particular, many management procedures are extremely important related to the excellent outcome of vaccination-animal density, environmental control, level of stress, cleanliness of the environment and drinking water.
Developing procedures for most animal vaccines still relies on a classical strategy with live pathogens that possess a strong immunogenicity either with high virulence or without virulence. However, over the decade there has been great acceleration in the advancement of modern molecular techniques and the compilation of genomic data of many pathogens. Such advances provide a great opportunity to create desirable vaccine strains which are less dangerous but more effectively immunogenic than those of vaccines achieved by classical methods. It is well established that the immune system has several effector mechanisms to cope with various pathogens, which would be dependent on their lifecycle and the microenvironment of the infected host. Since killed vaccines are still mainly used for livestock, it is absolutely necessary to develop novel adjuvants in order to enhance satisfactory immunity for such vaccines. Potent adjuvants should be able to effectively elicit cellular immunity in animals that are vaccinated with less immunogenic vaccines including killed or subunit vaccines. The other way to resolve this issue would be to develop a new delivery system, such as plasmid DNA, liposome, microparticles, and live viral or bacterial vectors, which can introduce vaccine antigens into intracellular compartments. Another notable advancement in immunology is the increased recognition on the major roles of innate immunity in vaccine adjuvant functions, which is often ignored despite their significant influence on vaccine developments. Recently discovered innate immunity receptors are screened as new adjuvant materials having activities, and they are used for inducing or enhancing vaccine reactions. Currently many types of adjuvants are in use for animal vaccines.
Commercialization of vaccine products should fulfill some regulations for vaccine efficacy, safety, and development processes instructed by governments. In the US, most animal vaccines come under United States Department of Agriculture (USDA) regulation. In Korea, animal vaccines are regulated under Animal Plant and Fisheries Quarantine and Inspection Agency. In the EU, regulations are under the control of the law of the EU. These regulatory agencies take account of faster and lower cost routes to registration than those of human vaccines. With consideration of the commercial market, overall demand of animal vaccines is steadily growing due to the fast increasing livestock population. Overall, along with less stringent regulatory requirements, research and development of animal vaccines would be the forefront of experimental trials of innovative techniques and commercial opportunity.
NDV vaccine strains show promise as a base from which to develop effective vaccines against pathogens that infect animals and humans. Most NDV-vectored vaccines used for poultry are bivalent and provide protective efficacy against virulent NDVs and several foreign pathogens. NDV-vectored poultry vaccines have been developed to provide protection against HPAIV (A/H5 and A/H7), IBDV, ILTV, IBV, and aMPV. Safe NDV-vectored vaccines have been developed as antigen delivery vaccines for veterinary and human use. Such vaccines express the foreign target antigen and induce robust immune responses at both the local and systemic level as shown with NDV-vectored veterinary vaccines in cattle/sheep (e.g., BHV-1, BEFV, RVFV, and VSV), dogs/cats (e.g., CDV and RV), pigs (e.g., NiV), and horses (e.g., WNV). NDV-vectored human vaccines currently under development aim to provide protection against HIV, HPIV-3, and RSV, newly emerging zoonotic viruses (e.g., HPAIV A/H5, SARS-CoV, EBOV, and NiV), and noncultivable human viruses (e.g., human papillomavirus, hepatitis C virus, and NoV). A primary vaccination by an NDV-vectored vaccine expressing a foreign protein can be effective but the efficacy of an updated or new vaccine based on the NDV vector may be reduced by pre-existing NDV antibodies, which could limit the continuous use of NDV vector vaccines in human.
In conclusion, NDV vaccine strains are attractive vectors that can be used to develop effective vaccines against pathogens that infect animals and/or humans; such vaccines are safe and efficient, and provide high levels of protective immunity, although there is a risk that previous vaccinations can reduce the efficacy of the vectored vaccine.
Macroscopic pneumonia in the strict control group (non-infected, non-vaccinated) was minimal (0.08 ± 0.15), and macroscopic lesions were detected in only 2 of the 6 pigs inoculated with only B. bronchiseptica (Bb/NV/NCh), with a group average of 1.8% of the lung affected (Figure 2). There was not a significant increase in the percentage of gross pneumonia in the NV/Ch group when compared to the control group, though the percentage of pigs in each group presenting with lesions was different (100 vs. 25%, respectively). Also, there was not a significant difference between NV/Ch and LAIV/Ch groups (p > 0.05). The average percentage of lung affected by lesions for the NV/Ch group was 4.1 ± 2.9 compared to 2.8 ± 4.3 for the LAIV/Ch group. There was an increase in the percentage of lung affected in the Bb/NV/Ch and Bb/LAIV/Ch groups when compared to the strict control group (p < 0.05), but not between the Bb/NV/Ch and Bb/LAIV/Ch groups (p > 0.05).
Microscopic lesions were either not present or minimal (limited to mild interstitial thickening) in the strict control group (NV/NCh; Figure 3A), as well as in all but 2 of the pigs inoculated with B. bronchiseptica alone (Bb/NV/NCh). The two Bb/NV/NCh pigs with microscopic changes had lesions consistent with chronic B. bronchiseptica pneumonia characterized by moderate thickening of the alveolar septa with fibrin and collagen, type II pneumocyte hyperplasia, and alveolar spaces variably filled with macrophages (30) (Figure 3B). Pigs inoculated with IAV alone (NV/Ch) had mild lesions consistent with IAV infection characterized primarily by suppurative bronchitis and bronchiolitis with epithelial necrosis and peribronchiolar lymphocytic infiltration (31) (Figure 3C). The presence and severity of interstitial pneumonia was minimal to mild in the NV/Ch group. The IAV-associated lesions were diminished in the vaccinated group (LAIV/Ch) when compared to the non-vaccinated group (NV/Ch). In particular the suppurative bronchitis or bronchiolitis with epithelial necrosis was reduced; however, there was peribronchiolar lymphocyte infiltration and bronchus associated lymphoid tissue (BALT) hyperplasia in the majority of LAIV/Ch pigs (Figure 3D).
Pigs that were infected with B. bronchiseptica and subsequently challenged with IAV (Bb/NV/Ch) had microscopic lesions consistent with both IAV infection as well as acute and chronic B. bronchiseptica pneumonia (Figure 3E). Influenza lesions included suppurative bronchitis and bronchiolitis with epithelial necrosis and submucosal lymphohistiocytic inflammation, as well as peribronchiolar lymphocytic infiltration. However, the suppurative bronchitis and bronchiolitis tended to be more severe than that observed in pigs infected with IAV alone, and furthermore alveoli were variably filled with neutrophils and macrophages with areas of alveolar epithelial necrosis, hemorrhage, and type II pneumocyte hyperplasia, which is consistent with acute Bordetellosis. In addition, in sections from some Bb/NV/Ch pigs there were areas consistent with chronic Bordetellosis characterized by interstitial pneumonia consisting of alveolar septal thickening with mononuclear cells as well as fibrin and collagen (Figure 3E).
Finally, as noted in the LAIV/Ch group, vaccinated pigs that had been infected with Bordetella and challenged with IAV (Bb/LAIV/Ch) had diminished bronchial and bronchiolar epithelial necrosis. However, these pigs had lesions consistent with both acute and chronic Bordetella pneumonia, including acute lesions consisting of alveoli and bronchioles that were variably filled with neutrophils and/or macrophages and alveoli with areas of epithelial necrosis, hemorrhage, and type II pneumocyte hyperplasia (Figure 3F). Sections from some of the pigs in Bb/LAIV/Ch group also contained chronic lesions of interstitial pneumonia consisting of alveolar septal thickening with mononuclear cells as well as fibrin and collagen, which is consistent with chronic B. bronchiseptica infection.
Teicoplanin, a glycopeptide antibiotic, and its derivatives potently inhibit the entry of EBOV-GP-pseudotyped viruses in various cell types. Studies on the antiviral mechanism indicated that teicoplanin blocks EBOV entry by specifically inhibiting the activity of cathepsin L, thus avoiding the maturation of GP and the release of the viral genome into the cytoplasm. The antibiotic azithromycin has been demonstrated to inhibit eVLP entry but further studies have not been performed and its mechanism of action is still largely uncharacterized.
Among CADs that have been proved to inhibit EBOV infection in screening experiments, there are also the antifungal drugs terconazole and triparanol, formerly used as cholesterol-lowering drugs, now withdrawn due to their numerous toxic side effects.
In the infected birds, clinical signs observed include white, soft-shelled eggs, greenish diarrhoea, and respiratory distress (coughing, sneezing, and rales). As at the time of sampling, egg production had dropped by approximately 55%. Mortality was observed before the birds came into lay and continued during lay with a range of 1%–2%. At post-mortem, lesions observed include cloudy air sacs, frothy and congested lungs, whitish, cheesy materials on the serosal surface of the intestine, white nodules on the surface of the ventricles, proventriculus and intestines, and presence of ascitic fluid in the abdomen. These signs and lesions are consistent with findings from previous reports (Awad et al., 2014b; Ballal et al., 2005). Due to the similarities in clinical and pathological lesions presented by infections involving respiratory viruses, clinician that based their diagnosis on these non-pathognomonic signs may miss the aetiological agents. Though the mortality rate in the affected flock was low (1%–2%), it is consistent with the report by Awad et al. (2014a). Generally, single infections with IBV result in low mortality. However, exacerbation by concurrent infection with other pathogens of viral or bacterial origin have been reported (Jackwood, 2012).
As shown in Figure 1, the tissue homogenate was positive for IBV by RT-PCR and negative for AIV and NDV. Upon inoculation of ECEs with the tissue homogenate, no noticeable changes were observed in the embryos in the first few passages. However, at passage four, embryo death with characteristic IB lesions, including curling, dwarfing, and hemorrhages on the embryos (13 d of age) were conspicuously discernible (Fig. 2). Allantoic fluids harvested from the eggs of both dead and live embryos did not cause agglutination of chicken red blood cells in spot hemagglutination test (data not shown) and this confirms the absence of hemagglutinating agent. In this study, we have shown that IBV which is less described and often given less attention and not NDV or AIV was the causative agent of infection in the 54-wk-old laying birds showing respiratory signs and severe drop in egg production. Although ND was first suspected by the consulting clinician due to its enzootic status in Nigeria. In a limited study, the prevalence of IB was found to equal that of ND confirming the increasing important enzootic status of IB in Nigeria poultry (Shittu unpublished data). In this study, successful isolation of IBV in embryonating eggs was accomplished after four blind passages with the embryos developing lesions characteristic of IB such as stunting and dwarfing (Fig. 2). For IBV isolation, ECE and tracheal organ cultures (TOC) are substrates of choice although TOC has an edge over ECE in that stasis of the tracheal cilia could be observed in the former upon primary inoculation (OIE, 2008). In this study, ECE isolation technique was found to be equally useful.
According to the farm records, the birds were vaccinated with inactivated oil-emulsion vaccine which contained IBV antigen. However, this seemed not to have protected the birds against morbidity, mortality, and decreased egg production. It has been reported that chickens with low antibody level to IBV serotype could experience severe drop in egg production, whereas those with high antibody level are less affected in terms of egg quality and production possibly as a result of immune protection (Ballal et al., 2005). Available literature show that the use of inactivated IB vaccines alone does not confer adequate protection on the birds except where they are first primed with live attenuated IB vaccines during the early stages of production (Cook et al., 2012). In addition, IBV serotypes do not cross-protect (Jackwood et al., 2010), thus the vaccine must be designed based on circulating serotypes in the locality. In Plateau State, there are no available data on the circulating IBV strains. Although most vaccines being used on the field in Nigeria are predominatly Mass serotype, detection of other serotypes in this investigation is a further indication and support speculation that the vaccine strains being used differ from some of the serotypes in circulation. Furthermore, Ducatez et al. (2009) identified a novel IBV serotype “IBADAN” from southwestern Nigeria and no information exists on the ability of the vaccine strains in use to protect birds against this novel strain (de Wit et al., 2010). It is, however, not known if this serotype circulates in the northern part of the country.
Antibody prevalence and high GMT titre distribution of the three serotypes of IBV used in the study for Mass, Conn, and Ark are 490.5, 215.3, and 534.9, respectively, as shown in Tables 2 and 3. In all 32 serum samples tested, 100% seropositivity was also observed for Mass, Conn, and Ark serotypes. In addition, concurrent infections with Mass/Conn, Mass/Ark, Conn/Ark, and Mass/Conn/Ark serotypes were observed.
Interestingly, the HI results for the three serotypes (Mass, Conn, and Ark) tested in this study revealed 100% seropositivity (Table 2). This clearly shows that the three IBV serotypes are present in the farm and may be in circulation in Plateau State with the possibility of other hitherto unreported serotypes. As reported by Jackwood (2012), several serotypes and variants of IBV circulate around the world with some having specificity for a particular location, making them indigenous to those places. Such may include the newly described serotype by Ducatez et al. (2009) which we could not test for in our samples due to unavailability of strain specific diagnostic reagent. The Mass strain of IBV has been reported to be widespread across the globe possibly due to its use as a vaccine (de Wit et al., 2010). In Nigeria, breeder stocks are often vaccinated with live IBV vaccine using Mass-like strains at much younger ages (Ducatez et al., 2009). However, in this case, a trivalent killed-adjuvanted vaccine containing IBV was said to have been administered without prior priming with live IBV vaccine. As previously reported (Bijlenga et al., 2004; de Wit et al., 2010), antibody response to killed-adjuvanted IBV vaccine without priming the birds with live attenuated IB vaccine are usually poor. It can, therefore, be deduced that the high titre of antibodies to the three IBV serotypes detected in this study (Table 3) may not have emanated from vaccination, but could be a result of recent or continuous infection with circulating strains of the virus as also shown by molecular detection and virus isolation. To the best of authors’ knowledge, co-circulation of multiple serotypes of IBV as described here is the first documented report from Nigeria.
Detection of antibodies to S. suis was made by an indirect ELISA designed for that purpose. The antigen was produced by cultivating S. suis (strain CCUG 4255) for 18 h at 37 °C on horse blood agar plates. From each plate, the whole growth was harvested in 2 ml PBS without Ca and Mg (pH 7.4; SVA art no 302800) and ultrasonicated (MSE, 60 W ultrasonic disintegrator, Measuring Scientific Equipment Ltd, London, UK) for 5 min per 8 ml solution at 1.3 Ampere with an amplitude of 10 µm. The ultrasonicated solution was centrifuged at 12,000g for 20 min at 4 °C (RC2B, Sorvall, Newton, USA). Thereafter, the liquid phase was collected and stored at −20 °C.
Each well in a microtiter plate (Greiner Bio-one, Sigma-Aldrich) was coated with 100 µL of the sonicated antigen diluted 1/10,000 in PBS-T in room temperature for 18 h. Thereafter the microtiter plate was washed three times with PBS-T, and 100 µL serum diluted 1/100 in PBS was added to duplicate wells and the plates were incubated at 37 °C for 1 h. The plates were again washed three times with PBS-T and 100 µL of the conjugate (Protein A-horseradish peroxidase conjugate, Bio-Rad, Richmond, USA) diluted 1/5000 with PBS-T was added to each well and the microtiter plates were stored for 1 h in 37 °C. Then the plates were again washed three times with PBS-T and 100 µL of the substrate with tetra methylbenzidine (TMB, SVANOVA Biotech, Uppsala, Sweden) was added to each well. The reaction was stopped with 100 µL H2SO4 after 10 min and the absorbance was read at 450 nm by a spectrophotometer (Multiscan MCC/340® MK type II, Labsystem OY, Helsinki, Finland). The results obtained were adjusted to A450 = 1.0 for a positive standard serum and absorbance values exceeding 0.5 were regarded as positive reactions, based on the mean absorbance value +2 standard deviations of samples from 72 pigs without clinical signs of S. suis infection (A450 = 0.26 ± 0.12).
This report underscores the need to investigate, by laboratory diagnostic methods, all cases presenting with respiratory distress and drop in egg production for IB. In the present case prior to further laboratory, most clinical diagnoses are based on signs and pathological lesions. Future observation and investigation should be designed to investigate the different IBV serotypes and genotypes in circulation across the country with the aim of producing vaccine (s), based on the identified serotypes, for combating the menace of IB in the Nigerian poultry population.
Rabbits (Oryctolagus cuniculus) are well known for their use in studying cardiovascular diseases, antibody production, and eye research. Rabbits were also employed to study pneumonia, although only a few models are available. Typical read-out parameters include survival, leukocyte infiltration of the lungs, lung pathology, and assessment of drug concentration in serum. One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz (1913). This study revealed that rabbits possess an active immunity if they have recovered from one attack of experimental pneumonia and they may subsequently resist repeated intra-tracheal dosages of pneumococci (Kline and Winternitz, 1913). In 1926 an infection by inhalation of Type I pneumococci was established in rabbits (Stillman and Branch, 1926). The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals (Stillman and Branch, 1926). Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis (Diep et al., 2010, 2017), as well as drug efficiency and efficacy studies (Cabellos et al., 1992; Croisier-Bertin et al., 2011). However, this infection route requires surgery and species-specific reagents are scarce. In IAV research rabbits are frequently used for antibody production and for studies on antibody kinetics following single or multiple IAV administrations (Loza-Tulimowska et al., 1977). Also, rabbits are used for safety investigations of vaccines (e.g., CoVaccine HT or Aflunov) (Heldens et al., 2010; Gasparini et al., 2012). In recent years the shedding of avian IAV by cottontails (Sylvilagus spp.) was investigated revealing that nasally and orally inoculated cottontails shed relatively large quantities of viral RNA (Root et al., 2014). Notably, low viral titers were found to be sufficient to initiate viral replication in cottontails (Root et al., 2017). However, despite their susceptibility to IAV infection, rabbits are only rarely used as model for IAV pathogenesis since they offer no improvement over other established infection models.
This study was carried out in accordance with the recommendations of the USDA-ARS-National Animal Disease Center Animal Care and Use Committee and the protocol was approved by the Committee.
Respiratory illness is traditionally regarded as the disease of the growing pig, and has historically been associated with bacterial infections such as Mycoplasma hyopneumoniae [1–3] and Actinobacillus pleuropneumoniae [4–6]. These bacteria still are of great importance, but the continuously increasing herd sizes have complicated the clinical picture. As the number of transmission events between pigs in a population is equal to the number of pigs multiplied with the number of pigs minus one [x = n * (n − 1)], they will escalate as the herd size increase. Thus, the number of transmission events between pigs will increase with a factor of around four if a population is doubled and with a factor of around 100 if a population is enlarged ten times.
The increased number of transmissions between pigs may increase the influence of other microbes. M. hyopneumoniae and A. pleuropneumoniae are important pathogenic microbes, but co-infections may intensify or prolong clinical signs of respiratory disease [8–11]. It has also been observed that the incidence of respiratory illness may vary with season. Therefore, infections in the respiratory tract of grower pigs have become regarded as a syndrome rather than linked to single microorganisms [11, 13, 14]. This syndrome is referred to as the porcine respiratory disease complex (PRDC). As stated above PRDC is regarded to be dominated by bacterial species, and important primarily pathogenic bacterial species include M. hyopneumoniae [1–3] and A. pleuropneumoniae [4–6]. The frequent demonstration of interferon-α in serum in growers during the first week after arrival to fattening herds [15, 16] suggest that PRDC can be associated with viral infections, and that PRDC can also include the influence of secondary invaders such as Pasteurella spp [17, 18].
When Sweden in 1986 as the first country in the world banned the use of low dose antibiotics in animal feed for growth promotion, some introductory health disturbances were recorded. As a consequence, a strict age segregated rearing from birth to slaughter was implemented in a large scale, which improved health as well as productivity [19, 20]. As seen in Fig. 1, the incidence of recorded pathogenic lesions in the respiratory tract at slaughter decreased during the last decade of the twentieth century. The registrations of pneumonia at slaughter has remained stable at that level since then. In contrast, the incidence of recorded pleuritis at slaughter has continuously increased since the year 2000, as has the clinical evidence of actinobacillosis. Discussions concerning the reason for this increase has included suggestions of introduction of new strains, or mutation of existing strains of A. pleuropneumoniae. However, acute actinobacillosis has in Sweden historically been dominated by serotype 2, and is still dominated by that serotype. Further, Pulse Field Gel Electrophoreses has revealed that strains isolated in the twenty-first century were identical to strains isolated in the 1970s and 1980s. As a consequence, the increase of actinobacillosis and pleuritic recordings at slaughter has merely been linked to the continuously increasing herd sizes with increasing number of transmissions of microbes between pigs, within and between units.
The aim of this study was to validate the presence of A. pleuropneumoniae and M. hyopneumoniae, as well as the secondary invaders P. multocida and Streptococcus suis in pig herds with a high incidence of pleuritic lesions at slaughter.
Bivalent NDV-vectored vaccines, which have been developed to prevent diseases of economic importance to the poultry industry, have advantages over traditional vaccines (Table 3). Examples include infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotrachitis virus (ILTV), and avian metapneumovirus (aMPV).
IBDV, a birnavirus that infects chickens, is an important pathogen that causes severe immunosuppression and high mortality in young chickens. Live attenuated vaccines of moderate virulence (especially widely used intermediate plus vaccines) are used widely to prevent infectious bursal disease (IBD); however, they can cause severe side effects (symptoms consistent with IBD) in young chickens. Huang et al. developed a NDV-vectored IBDV vaccine (rLaSota/VP2) expressing the VP2 gene of IBDV, which is responsible for protective immunity against IBDV. The VP2 gene is inserted into the 3'-end non-coding region of the NDV genome. The live IBV vaccine is very safe in young chickens and protects SPF chickens against virulent NDV and virulent IBDV.
IBV, a coronavirus that infects birds, causes respiratory disease and renal disorders (the nephropathogenic strain) in poultry and poor egg production in laying hens worldwide. Currently available live attenuated IBV vaccines risk giving rise to new variants through recombination with field IBVs. This often reduces the efficacy of IBV vaccines. Importantly, live IBV vaccines interfere with the live attenuated NDV vaccine. To overcome the limitations of currently available live vaccines, Toro et al. developed a NDV-vectored IBV vaccine (rLS/IBV.S2) expressing the S2 subunit of the IBV S glycoprotein. Oculo-nasal immunization of chickens (1.0×107 EID50/dose) provided complete protection from clinical disease (mortality) after challenge with a lethal dose of virulent NDV (CA02). The protective efficacy of the rLS/IBV.S2 vaccine was also assessed using a heterotypic protection approach based on priming with a live attenuated IBV Mass-type vaccine followed by boosting with rLS/IBV.S2. The vaccine protected chickens against clinical disease after lethal challenge with a virulent Ark-type IBV strain, leading to a significant reduction in virus shedding when compared with that in unvaccinated/challenged chickens.
ILTV, a herpesvirus that infects birds, causes respiratory disease in chickens. Currently available live attenuated ILTV vaccines are effective, but there are concerns about safety in chickens because of the risks of virulence acquirement and latent infections during bird to bird transmission. Bivalent NDV-vectored vaccines against ILTV have been developed to overcome side effects associated with the live ILTV vaccine. Kanabagatte Basavarajappa et al. developed a NDV-vectored ILTV vaccine (rNDV gD) expressing glycoprotein D (gD) of ILTV. The protective efficacy of the rNDV gD vaccine against challenge with virulent ILTV and virulent NDV was then evaluated in SPF chickens. Immunizing chickens with rNDV gD (106 TCID50/dose) via the oro-nasal route induced a strong antibody response and provided a high level of protection against subsequent challenge with virulent ILTV and NDV, indicating that rNDV gD has potential as a bivalent vaccine.
Outbreaks of Ad in the general population have been characterized by infection due to novel viruses such as Ad7h, Ad7d2, Ad14a, and Ad3 variants. These novel viruses are sometimes associated with high attack rates and a high prevalence of pneumonia. Severe mortality is also prevalent among patients with chronic disease and in the elderly.
One of the most important novel serotypes, Ad14, previously rarely reported, is now considered as an emerging Ad type causing severe and sometimes fatal respiratory illness in patients of all ages (45). Beginning in 2005, Ad14 cases were suddenly identified in four locations across USA (46); the strain associated with this outbreak was different than the original Ad14 strain isolated in 1950s. The novel strain, Ad14a, has now spread to numerous US states and is associated with a higher rate of severe illness when compared to other Ad strains.
Novel Ad species have also been recently detected in cross-species infections from non-human primates to man in USA and between psittacine birds and man in China (47). These cross-species infections indicate that Ads should be monitored for their potential to cause cross-species outbreaks. In a recent review of the risks of potential outbreaks associated with zoonotic Ad (48), it was noted that intense human–animal interaction is likely to increase the probability of emergent cross-species Ad infection. Additionally, the recombination of AdVs with latent “host-specific” AdVs is the most likely scenario for adaptation to a new host, either human or animal.
Currently, there are no FDA approved antivirals for Ad infection; however, the best antiviral success has been seen with ribavirin, cidofovir, and most recently brincidofovir an analog of cidofovir (49).
Swine influenza is a highly infectious acute respiratory viral disease of pigs that affects the respiratory tract and has considerable economic impacts. Three main subtypes of swine influenza virus (H1N1, H3N2, and H1N2), with H1N1 as the predominant subtype, have circulated in pigs worldwide [2, 3]. In March 2009, a new swine-origin H1N1 influenza virus became a pandemic. Pig infections with the new H1N1 virus have then been observed in multiple countries, showing that the pandemic H1N1 viruses have become established in swine populations [5–7]. Previous study has showed the new H1N1 viruses have spread from humans to pigs in China. Swine influenza virus replication is mainly restricted to the epithelial cells in the respiratory tract, with the lung being the major target organ. Although it is a highly contagious virus for pigs and has high-morbidity but low-mortality rates, secondary complications would substantially worsen the illness and increase death rate. In fact, swine influenza is one of the several significant contributors to the porcine respiratory disease complex (PRDC), which is caused by infection with more than one pathogen, such as the swine influenza virus and Streptococcus suis (S. suis) co-infection.
S. suis infections have been considered as a major problem worldwide in the swine industry and as a secondary agent of pneumonia, particularly in the past 20 years. Among the 35 serotypes, serotype 2 (SS2) is generally considered as the most prevalent and virulent type. S. suis infections in pigs often cause arthritis, meningitis, pneumonia, endocarditis, and septicemia with or without sudden death. Although S. suis is a major swine pathogen, it has been increasingly detected in wide range of mammalian species. Infections have been observed in humans in 2005 in China, which affected more than 200 people and had approximately 20% mortality. In clinical cases, co-infections of swine influenza virus and S. suis in pigs often contribute to severe pneumonia and can increase the mortality. Co-infection outbreaks have been recently reported in England.
Recently, several studies on the pathogenesis of the co-infection of influenza virus and Streptococcus pneumonia have been performed using mouse models [13–15]. Pro- and anti-inflammatory (IL-6, IL-1β, TNF-α, and IL-10) molecules were remarkably elevated in the blood in influenza virus and Streptococcus pneumonia co-infected mouse. However, fewer studies have examined swine influenza and S. suis co-infection in pigs, and its pathogenesis is not yet fully elucidated. In the present study, microarray assay was utilized to explore the global host responses of porcine lungs that suffered from H1N1 influenza virus, SS2, H1N1-SS2 co-infection, and phosphate-buffered saline (PBS) to enhance the understanding of the H1N1 and S. suis co-infection pathogenesis through a pig model. Stronger inflammatory and apoptosis responses were determined to be important contributors to the increased pathogenicity caused by swine H1N1 and SS2 co-infection. Our study would improve the understanding of the pathogenesis of H1N1 and SS2 co-infection in pigs.
First, 600 µl per well of basolateral supernatants from JEV-infected porcine NEC were harvested at 72 hpi and dispensed into 24-well plates. Next, 106 monocytes were added into Transwell inserts with 3-µm-diameter pores (Becton, Dickinson) and placed in the wells filled with the porcine NEC culture-derived basolateral medium. After 3 h of incubation at 39°C and 5% CO2, basolateral medium was harvested and total monocytes were harvested and quantified. Monocyte phenotype was verified by immunolabeling with anti-porcine CD14 (MIL2; Bio-Rad). Controls included ALI medium, medium collected from mock-treated porcine NEC, and porcine CCL2 (200 pg/ml in ALI medium; Kingfisher Biotech, St. Paul, MN). For monocyte quantification by flow cytometry, CountBright absolute counting beads (Thermo Fisher) were used following the manufacturer’s manual. Dead cells were excluded by electronic gating in forward/side scatter plots, followed by exclusion of doublets.
APP serotype 2 was the most common cause for acute respiratory outbreaks in finishing pigs in Finland and A. suum or other opportunistic bacteria caused acute coughing episodes in some herds. Viral pathogens appeared to have a minor role in causing the clinical signs. Field necropsies supplemented with microbiological analysis were the most valuable diagnostic tool combination in detecting the main cause of the infections under field conditions. Bacterial isolation from the lungs was especially important in assessing antimicrobial susceptibility and for optimizing antimicrobial treatment, because some resistance, especially to tetracycline, was found among the APP strains causing disease. Serological diagnostics were not optimal in the diagnosis of the respiratory outbreaks of our study. Although several different diagnostic methods were used, the primary pathogen causing the outbreak remained questionable in some herds.
The breadth and depth of arbovirus surveillance differs regionally, and several areas lack surveillance altogether. There is also a lack of inter-disciplinary expertise on arbovirus diseases and understanding their vectors, and epidemiology. In addition, only a small number of arboviral diseases can be prevented using vaccines or specific antiviral drugs, and there are few validated diagnostic reagents, with which to monitor disease progress and control. Until such disease control reagents become available, the most effective alternative is to focus on practical procedures to reduce risks of exposure to arthropods.
Respiratory infections in pigs are very important factor affecting the profitability of pig production [1, 2]. Although various bacteria or viruses could induce the respiratory infection separately, it has commonly been caused by coinfection with more pathogens under field conditions [1–3]. The most important infectious agents responsible for infection of the respiratory tract in pigs are: swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV), Pasteurella multocida (Pm), Actinobacillus pleuropneumoniae and Mycoplasma hyopneumoniae [2, 4–6]. Besides, the above mentioned pathogens, the Haemophilus parasuis (Hps) can also be recovered from the lungs of pigs with pneumonia [1, 7–10]. In these cases Hps is often isolated along with other bacterial or viral pathogens and, therefore, the role of Hps in producing pneumonia is not clear [8, 11].
Bacterial pneumonia secondary to influenza is often observed in pigs. SIV is a significant contributor to the respiratory diseases and may predispose to secondary bacterial infection. Hps is an important and common respiratory pathogen of pigs. It can be a primary pathogen or be associated with other diseases such as SIV [3, 8]. It could be also isolated from nasal cavity, tonsils and trachea of apparently healthy pigs [8, 14]. Under favorable conditions, Hps can cause severe systemic infection characterized by fibrinous polyserositis, arthritis and meningitis [8, 11, 14]. Factors leading to systemic infection by Hps have not been clarified to date [9, 14].
Although there are previous reports of experimental reproduction of Hps or SIV infection in conventional pigs, little is known about the effect of concurrent infection with SIV and Hps on the disease severity and inflammatory response in pigs, even if this coinfection is common under field conditions [13, 15–17]. There are also limited data on the role of Hps in the production of pneumonia in the absence of other respiratory pathogens. Furthermore, the kinetics of acute phase protein (APP) response in SIV/Hps co-infected pigs has not been studied to date. As it has been shown for other pathogens, the exposure to several pathogens can lead to a stronger APP response, as compare to single infection [18–20]. Thus, in order to investigate the influence of SIV and Hps coinfection on clinical outcome, both local and systemic inflammatory response as well as pathogen shedding and load at various time points following intranasal inoculation, three experimental infections (Hps- and SIV-single infection, SIV/Hps co-infection) has been performed in the present study. The correlation between local concentration of cytokines and severity of infection (clinical score, lung score) as well as serum APP concentration has been also studied.
Porcine nasal mucosa tissue slices 4 µm thick were washed in confocal buffer (CB; 50 nM ammonium chloride and 0.1% saponin in PBS−/−) for 30 min and then incubated with anti-flavivirus group antigen antibody 4G2 for 2 h. After 3 washes with CB, the tissue was incubated for 1 h with Alexa Fluor 488-coupled anti-IgG2a (Thermo Fisher), and Cy3-conjugated anti-β-tubulin (Abcam, Cambridge, UK) was used for the detection of cilia. Porcine NEC cultured on the Transwell membranes were washed with PBS−/− and incubated in 4% PFA for 15 min. In the second step, the membranes were incubated in CB for 30 min. Cells were incubated with anti-ZO-1 (Thermo Fisher) and anti-flavivirus group antigen antibody 4G2 for 2 h, followed by Alexa Fluor 633-coupled goat anti-rabbit IgG (Thermo Fisher), Alexa Fluor 488-coupled anti-IgG2a (Thermo Fisher), and Cy3-conjugated anti-β-tubulin (Abcam). For evaluation of cell death in porcine NEC, Abcam’s Fixable Cell Viability Assay Kit (Fluorometric-Blue)-CytoPainter was applied for 30 min, and then cells were washed with PBS−/− and fixed with 4% PFA, followed by an incubation with anti-flavivirus group antigen antibody 4G2 as described before and labeling with phalloidin conjugated with AF555 (Thermo Fisher) and Alexa Fluor 488-coupled anti-IgG2a (Thermo Fisher) for 1 h. As a positive control for cleaved caspase 3 (C-Cas3) staining, apoptosis in porcine NEC was induced by incubation with anti-human CD90 (5E10; Becton, Dickinson). For detection of apoptotic cells, inserts were fixed with 4% PFA and incubated in CB for 30 min, incubated with anti-C-Cas3 (Cell Signaling Technologies), anti-flavivirus E protein antibody 4G2 for 2 h, washed, and labeled with phalloidin conjugated with AF647, Alexa Fluor 633-coupled goat anti-rabbit IgG, and Alexa Fluor 488-coupled anti-IgG2a (Thermo Fisher) for 1 h. All incubations were performed at room temperature in the dark. For each staining, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) at 2 µg/ml was applied for 5 min at 37°C and washed off with PBS−/−. Then tissue slices of porcine NEC were mounted on glass slides in Mowiol 4-88 reagent (Sigma). For confocal microscopy analysis, a confocal microscope A1 (Nikon AG) combined with an ECLIPSE Ti inverted microscope (Nikon) and digital imaging Nikon software (AR 3.30.02) were used. The image acquisitions were performed with the 40× objective, sequential channel acquisition and not simultaneous was employed; in order to give high-resolution images, the acquiring setting was performed with optimized voxel size and automatic threshold. The images were analyzed with Imaris 8.0.2 software (Bitplane AG, Zurich, Switzerland). To avoid false-positive emissions, different settings were applied, including background subtraction, threshold applications, gamma correction, and maxima. Insert surface staining (for E protein, CytoPainter, and C-Cas3) was calculated as average of positive signal of each channel from 20 different nonoverlapping fields in triplicate porcine NECs using confocal microscopy.