We here describe that transmission of SIV to humans, though rare, can occur and cause severe disease requiring life support through ECMO. Monitoring of people in direct contact and not wearing personal protective equipment revealed no secondary cases.

Rhinoviruses are small, single-stranded RNA viruses in the picornavirus family that are responsible for more than half of all upper respiratory tract infections. In addition to exacerbating asthma and chronic obstructive pulmonary disease, rhinoviruses have also been associated with acute respiratory hospitalizations among children (30). In a large prospective study of US pneumonias, rhinoviruses have been identified as the second most prevalent etiology of pneumonia in children after respiratory syncytial virus and the first most common etiology among adults (31). There are more than 150 unique types of rhinoviruses. Among the three genotypes (A, B, and C) types A and C are most often associated with increased morbidity and bacterial secondary infection. In animals, rhinovirus type C has been associated with morbidity in chimpanzees (32). With an array of unique serotypes no vaccines or approved antiviral therapies have been commercially produced; however, experiments have suggested that vaccines and antiviral therapy may be possible (33, 34).

First discovered in 1953 by Rowe et al., Ads are non-enveloped, double-stranded DNA viruses with 57 unique serotypes, some of which are specific for attacking the respiratory track, conjunctiva, or gastrointestinal track (40). Key features of Ad infections include various symptoms of disease, including rhinorrhea, nasal congestion, cough, sneezing, pharyngitis, keratoconjunctivitis, pneumonia, meningitis, gastroenteritis, cystitis, and encephalitis. Illnesses may be asymptomatic, mild, or severe; however, immunocompromised patients and infants are at increased risk of severe morbidity and death.

Actinobacillus pleuropneumoniae was the most common cause of acute respiratory outbreaks in the studied finishing pig herds. However, SIV, Ascaris suum, PCV2 and certain opportunistic bacteria appeared to cause concurrent infections, potentially contributing to the respiratory disease outcome. SIV and PCV2 were detected also in control herds suggesting possible subclinical infections in these herds. Serology alone was not effective in determining the cause of a respiratory outbreak, but pathology and bacteriology were considered useful in reaching a complete diagnosis. In addition to this, bacteriology together with antibiotic resistance determination was valuable in selecting the correct medication to be used, which is important in practice.

Clinical examination of the case herd pigs revealed respiratory signs including higher rectal temperature and coughing when compared to the control herd pigs. We were unfortunately unable to acquire exact data on the mortality in the rooms, because some animals in several herds were moved before or between herd visits to other rooms housing sick animals. However, the clinical signs in the herds were quite mild compared to those reported in experimental studies. During the first herd visit, the average rectal temperature of the case pigs was 39.7 °C and by the second herd visit it was at the same level as in the control herds. In a study by Loeffen et al., pigs in 15 respiratory outbreaks, caused by similar pathogens as in our study, showed respiratory symptoms with fever rising to 40–42 °C, which is much higher than the body temperature measured in our study. We cannot rule out missing the peak body temperature in our study pigs, as this might have happened earlier than our first herd visit. In addition to coughing, sneezing was very commonly heard in our study herds. However, sneezing was also diagnosed frequently in the control herds and its occurrence did not decrease by the second herd visit, indicating a persistent cause present in all herd types.

APP2 was the main causative agent for acute respiratory infections in Finnish finishing pigs. Previous Finnish studies screening the APP serotypes present in the country have revealed that APP2 is a common serotype together with several others [23, 24]. However, these older studies detected only antibody prevalence, but did not establish the connection between antibody prevalence and clinical disease. Our present study considered APP2 to be the main etiological agent of respiratory outbreak in the majority of herds (14 out of 20). In addition, APP2 was isolated from two other herds: one herd with miscellaneous etiology of its respiratory outbreak and one herd with lung lesions caused by A. suum.

It is difficult to compare the role of APP in respiratory infections in various countries, as study herds have usually not been selected and sampled in the same way as in our study. A study on clinical outbreaks carried out in the Netherlands in a manner similar to ours found APP to be the most likely cause in five out of 16 clinical outbreaks. Other researchers have often used slaughterhouse data and/or serology, but pathological findings from samples taken during visible clinical symptoms have not been used. This is most likely due to the difficulty in carrying out such field studies. In France, a cross-sectional study on infectious agents in respiratory diseases was performed in 125 French swine herds without including any information concerning the clinical situation of the herds. The researchers found that APP2 (serological diagnosis) was significantly associated with extensive pleuritis in the slaughterhouses, but not with pneumonia. In addition, an association between pneumonia or pleuritis in the slaughterhouses and seropositivity to APP was found in three other studies [26–28].

Serology has indeed been used in several studies examining APP causing respiratory infections in finishing pigs, but serological results have usually not been connected to clinical findings or acute outbreaks. Herds e.g. in Spain, Italy, Canada and Belgium have commonly been found positive for APP antibodies. In our study, antibodies against ApxIV toxin and APP2 LPS were found in both the case and control herds already during the first herd visit. Detectable antibody levels have been reported 1–3 weeks after experimental infection. Pigs in our study may have been in contact with APP long enough to enable some of them to have seroconverted earlier than the first herd visit. We know that the herd owners waited for an average of 8 days before communicating about a respiratory outbreak, which is quite a long time. Estimating the role of subclinical infections is also difficult. Subclinical APP infection is known to potentially cause seroconversion. A subclinical APP infection was possibly ongoing in the control herds during the time of our study and the presence of an infection went unnoticed by the herd owner and the research personnel. Our study found that the use of serology in APP detection, in either single sampling or paired samples, for the diagnosis of acute respiratory disease in field conditions is of little value, because no exact information of the initiation time of the infection is available and because of subclinical infections. However, when the beginning of an infection is known, as is often the case in experimental studies, or when the course of the infection is followed, serology remains a valuable diagnostic tool.

Three out of 20 outbreaks had a miscellaneous etiology. The pathology and bacteriology of these herds revealed findings incompatible with the set criteria for acute APP or SIV infections and the presence of bacteria such as APP, E. coli, P. multocida, S. aureus, Actinomyces spp., S. dysgalactiae subs. Equisimilis, S. suis and Streptococcus spp. APP serology showed seroconversion in each of these herds. SIV seroconversion additionally happened in one herd. Other researchers have also found that reaching a proper diagnosis is not always easy under field conditions despite using several diagnostic methods. Researchers studying respiratory outbreaks in 16 herds in the Netherlands could not reach a definitive diagnosis in four herds. They concluded that secondary bacteria might have played a role in the clinical outbreaks where no evident cause could be found. It is also possible the primary pathogen could not be identified in our study, despite the utilization of several diagnostic methods.

Ascaris suum infection was found to be the main cause of respiratory clinical signs in three case herds (15%). At least in Finland this pathogen has been considered a minor disease agent, especially in modern management systems with concrete floors and without outdoor access. It is widely known that A. suum can cause verminous pneumonia, as the larvae migrate through the lung tissue during their lifecycle. Also, slaughterhouse statistics from Finnish yearly figures of approximately 2 million slaughter pigs confirm the importance of A. suum infestations (Finnish Food Safety Authority, personal communication). Liver condemnations due to milk spots caused by A. suum were recorded in an average 6.5% of the finishing pigs slaughtered during the years 2010–2015. Other, more specific diagnostic methods for ascarids, have shown the prevalence of this parasite to be high. For example, antibodies against A. suum were observed in 39% of the study herds in a Danish study on finishing pigs. A higher prevalence was found in Serbia when using the flotation method, with approximately 50% of swine herds being A. suum positive. Ascariasis is a clinically relevant disease, as it can cause production losses and impair the immunity achieved by vaccinations. Also, proper diagnosis is of utmost importance. The administration of antimicrobial agents is useless as a treatment method against ascariasis.

Viral pathogens appeared to be less important as a cause of acute respiratory symptoms in finishing pigs in our study. The significance of SIV infection varies in other studies. For example, a clinical field study in the Netherlands showed SIV to be the most frequent main cause of a clinical outbreak in 16 herds. In a recent study in Brazil, nearly 70% of the nasal swab samples taken from piglets expressing signs of respiratory disease were PCR-positive for SIV. Furthermore, SIV was the most common finding in the virological evaluations of diseased animals showing lung lesions. However, studies also exist in which SIV is detected from pigs suffering from respiratory symptoms or slaughtered finishing pigs with lung lesions, but other pathogens are more frequently observed [6, 8, 39]. Typically for SIV, the virus is often detected in combination with other pathogens [6, 38]. In our present study, SIV was not found in the nasal swabs or lung samples and none of the case herds were therefore classified to suffer from an acute respiratory infection caused by SIV. However, results from the nasal swabs might be at least partly false-negative because our nasal swab sampling took place fairly late compared to optimal timing. Herds were visited approximately 8 days after clinical signs commenced and, therefore, it might be more correct to designate case herds as suffering from sub-acute respiratory disease instead of acute respiratory disease, especially in the case of SIV infection. Nasal swabs should be taken within 4 days after infection onset to attain the optimal detection of SIV. Serology revealed that three case herds out of 20 appeared to have had a concurrent SIV infection. Two out of the eight control herds also had pigs that had seroconverted and possibly suffered from subclinical SIV infection. In addition, both the case and control herds had antibodies already during the first herd visit. SIV serology, similarly to APP serology, should be understood more as a monitoring tool rather than as a diagnostic one. Nowadays, a convenient diagnostic sample is oral fluid, since at a population level, the presence of a pathogen may be detected for a longer period.

PCV2 has been associated with several disease syndromes collectively named porcine circovirus diseases. The role PCV2 plays in PRDC has been suggested to always involve interaction or synergism with other respiratory pathogens. The proportion of PCV2-positive animals was similar in the case and control farms of our study. PCV2 is a ubiquitous virus and hence PCR-positive animals occurring on a farm is very likely irrespective of the farm’s disease status. When blood sample analysis is based only on a standard PCR method (positive and negative, but no quantification of viral counts) without histopathology or detection of the virus in lymphoid tissues, the method is not sufficient for establishing the actual role of PCV2 in clinical disease. However, we did not see compatible PCV2 gross pathology during the field autopsies carried out on the case farms. Based on the lack of typical circovirus gross pathology and no difference in the proportion of PCR-positive animals between the case and control farms, we concluded that PCV2 probably did not play a major role in acute respiratory disease detected on the case farms despite the pathogen being present on farms. Vaccinating pigs against PCV2 infections is a very common preventive measure in Finland, and this has most likely contributed to the low occurrence of the pathogen.

According to our hypothesis, certain pathogens causing acute respiratory symptoms in pigs, namely Mhyo, PRRSV and PRCV, were not found in the studied Finnish herds. Especially Mhyo and PRRSV are important pathogens involved in respiratory infections in many pig-producing countries despite PRRSV not being detected in certain countries e.g. Finland [6, 8, 24, 43]. Certain PRCV strains can also contribute to respiratory disease. The lack of these pathogens as causative agents of respiratory outbreaks in Finland makes the situation of finishing pig herds quite different and favourable compared to respiratory disease scenarios in several countries located across the world. The absence of these pathogens may have a significant impact on the prevalence of other respiratory pathogens. However, the similar disease situation is present also in specific pathogen free herds in other countries than Finland, and these farms and their veterinarians might benefit from obtained results.

The vaccination history of study animals may have had some influence on serological results. We do not know the exact vaccination scheme utilized for study animals. However, we know that at the time of the study, Finnish sow herds generally vaccinated all sows against erysipelas, parvovirus and colibacillosis and a vaccination of piglets against PCV2 is very common in the country. We also know that very few (most likely none of the herds in our study) herds vaccinated against APP or SIV. Based on that estimation, it is unlikely that vaccinations would have had any significant effect on APP serology.

In our present study, APP strains were susceptible to most of the tested antimicrobials. Only tetracycline resistance was detected in more than 10% of the isolates. Similar results have been found in other European countries. Thus, resistance to tetracycline among porcine APP is a growing problem. Other studies have occasionally observed resistance to penicillin, ampicillin and trimethoprim/sulfamethoxazole [45–49]. The Ministry of Agriculture and Forestry in Finland issued its first national recommendation for prudent antibiotic use in animals as early as 1996. Currently, the first-choice antimicrobial agent recommended in APP infections is G-penicillin and the second choice is tiamulin or tetracycline. It is notable that a few isolates were found to be resistant also to penicillin, ampicillin and trimethoprim/sulfamethoxazole, which further emphasizes the need to investigate the resistance of APP strains when selecting the appropriate treatment.

From a practical standpoint, the field necropsies supplemented with microbiological analysis was the most valuable diagnostic tool combination for detecting the main cause of acute respiratory infections in our study. Field necropsies have several advantages: the technique is simple, inexpensive and not pathogen-specific, preliminary results are promptly available and antimicrobial susceptibility results can be obtained from bacteria isolated from lesions. However, field necropsies are disadvantageous, since if acute disease is not leading to mortality, euthanasia should be performed. In acute respiratory outbreaks, field necropsies, sample-taking and antimicrobial susceptibility testing are extremely important, because resistance to certain recommended antimicrobials does exist. Susceptibility testing is necessary not only from the field veterinarian’s and single pig herd’s point of view, but also from a national policymaker’s perspective. Serology cannot be used alone in diagnosis, but offers detailed information about possible pathogens causing mainly subclinical infections. Also other diagnostic methods could be used. In addition to the already mentioned oral fluids, tracheobronchial swabs or lavage would be of help. Their disadvantage is the need for special equipment and/or sedation of the pig, which might limit this sampling method under field conditions.

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.

Pleuritic lesions registered at slaughter ranged from 20.5 to 33.1 % in the four herds. High levels of serum antibodies to A. pleuropneumoniae and P. multocida, either alone or in combination, were seen. Pigs in this study seroconverted to M. hyopneumoniae late during the rearing period (herd B–D), or not at all (herd A), confirming a positive effect of age segregated rearing in preventing or delaying infections with M. hyopneumoniae. The results obtained highlight the necessity of diagnostic investigations to define the true disease pattern in herds with a high incidence of pleuritic lesions.

There is increasing evidence that the nasopharyngeal microbiota plays an important role in the pathogenesis of acute viral respiratory infections (Teo et al., 2015; de Steenhuijsen Piters et al., 2016; Rosas-Salazar et al., 2016a,b). Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections (Tregoning and Schwarze, 2010). Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens. All of them are frequent colonizers of the human nasopharynx and they share many features including pathogenic mechanisms and clinical aspects (Figure 1). However, they also have unique properties.

Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin (Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009). They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia. In the last decades, the pathogen became resistant to an increasing number of antibiotics and methicillin-resistant S. aureus (MRSA) is now a major cause of hospital acquired infections (Hartman and Tomasz, 1984; Ubukata et al., 1989; Zetola et al., 2005). Also the rise of community-acquired S. aureus strains is of special concern, because certain clones are associated with very severe infections (Rasigade et al., 2010). Recent prospective studies demonstrated an increase in proportion of community-acquired methicillin-sensitive S. aureus in severe pneumonia cases (McCaskill et al., 2007; Sicot et al., 2013).

The pneumococcus is a typical colonizer of the human nasopharynx. About 20–50% of healthy children and 8–30% of healthy adults are asymptomatically colonized (McCullers, 2006). Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including community-acquired pneumonia, bacteraemia, and meningitis (Bogaert et al., 2004; Valles et al., 2016). This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly (Black et al., 2010; Valles et al., 2016).

Group A streptococci colonize the mouth and upper respiratory tract in about 2–5% of world’s population (Okumura and Nizet, 2014). The most common, non-invasive and mild infections caused by GAS are tonsillitis and pharyngitis with estimated 600 million cases per year (Carapetis et al., 2005). Listed as number nine in the list of global killers with around 500,000 deaths annually (Carapetis et al., 2005), it is obvious that this pathogen can cause severe invasive infections, including pneumonia, sepsis, streptococcal toxic shock syndrome, and necrotizing skin infections (Cunningham, 2000; Carapetis et al., 2005).

Although all three pathogens are able to cause highly lethal diseases, the most fatal remains the pneumococcus, estimated to cause ca. 10% of all deaths in children below 5 years of age (O’Brien et al., 2009), in the elderly (Marrie et al., 2017), and in immuno-compromised individuals (Baxter et al., 2016).

Recently, the cotton rat (Sigmodon hispidus) was reported to be susceptible to IAV. Nasal and pulmonary infection in adult inbred cotton rats did not require viral adaptation (Ottolini et al., 2005). The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days. Tissue pathology included damage of bronchiolar epithelium and the animals developed pneumonia which persisted for nearly 3 weeks (Ottolini et al., 2005). In bacteriological studies rats are more frequently used. There are numerous rat models investigating the impact of diabetes (Oliveira et al., 2016), metabolic syndromes (Feng et al., 2015), cirrhosis (Preheim et al., 1991), pharmaco-kinetics and dynamics (Antonopoulou et al., 2015; Hoover et al., 2015), intoxication (Davis et al., 1991), immunization (Iinuma and Okinaga, 1989), and general bacterial virulence factors (Shanley et al., 1996) on development of pneumococcal, streptococcal, and staphylococcal pneumonia and lung pathology. Unfortunately, there are only few studies on bacterial and viral co-infections in rats. The first was performed by Harford et al., 1946 (Harford et al., 1946). The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome (Harford et al., 1946). Another study on human respiratory syncytial virus and S. pneumoniae revealed that rats were easily colonized with pneumococci, but viral replication after subsequent infection was strain dependent. In addition, neither pneumococci nor the virus spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates (Nguyen et al., 2015). Although rats share a lot of immune features with humans, including nitric oxide production by macrophages (Carsillo et al., 2009), the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents.

Average rectal temperatures of the pigs during the first herd visit were 39.7 °C (SD 0.3, N = 448) and 39.4 °C (SD 0.3, N = 160) in the case and control herds, respectively (p = 0.01). Corresponding figures for the second herd visit were 39.3 °C (SD 0.1, N = 427) and 39.3 °C (SD 0.2, N = 155) (p = 0.3) for the case and control farms, respectively.

An average 4.0 (SD 3.8, N = 17, case herds) and 0.2 (SD 0.3, N = 8, control herds) coughing episodes were counted per 100 pigs during the first herd visit. The incidence rate ratio (IRR) for coughing episodes (case vs. control herds) was 16.5 (p < 0.01) during the first herd visit. By the second visit the coughing episodes in the case herds decreased to the same level as in the control herds: 0.6 (SD 0.8) for the case rooms and 0.4 (SD 0.5) for the control rooms. No difference in IRR was observed for the coughing episodes during the second herd visit (IRR 1.5, p = 0.5).

During the first herd visit, case pigs averaged 12.2 sneezing episodes per 100 pigs (SD 11.1) and the control pigs averaged 5.5 (SD 5.3). The IRR for sneezing episodes (case vs. control herds) was 1.9 (p = 0.1) during the first herd visit. By the second visit, the sneezing episode count in the case herds had decreased down to the same frequency as in the control herds: 5.5 (SD 4.4) in the case herds and 3.9 episodes per 100 pigs in the control herds (SD 3.3; p = 0.2).

Of the general population group (n = 186), 5% had dromedaries around the home, 3% had direct, recurrent contact with dromedaries, 18% frequently consumed raw camel meat and 22% consumed unpasteurised camel milk more than once per week .

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.

This study shows the clinical impact of acute infection with pandemic influenza (H1N1) 2009 virus in a naïve pig population. Typical signs of influenza-like illness and/or increased reproductive disturbances were reported from 40% of herds where infection with pandemic influenza (H1N1) 2009 has been documented. Clinical signs were reported from all age groups of animals. The proportion of animals affected, duration, and type of clinical signs varied between herds. Further studies are needed to investigate the reported reproductive disturbances in sows and to evaluate the economic impact of pandemic influenza A (2009) virus infection in the Norwegian pig population.

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.

This study shows that 40% of positive nucleus and multiplier herds reported clinical signs of pig ILI and/or increased reproductive disturbances. The low morbidity is surprising as one might expect higher morbidity rate given the naïve population and the nature of the disease. The low morbidity, however, corresponds well with another study carried out by the Norwegian Pig Health Service (personal communication Anne Jørgensen) where 51% of infected herds (including non-breeding herds) reported clinical signs. The high health status of pigs in Norway could have resulted in the lower morbidity, as some herds might have experienced subclinical infection or mild disease that was not registered nor reported by the farmer. Farmers were chosen as respondents in this study because they are more likely to have the most complete observations of an influenza outbreak occurring in their farm. While veterinarians are undoubtedly more qualified to perform clinical examinations and evaluations, they normally spend a limited amount of time on each farm, and typically do not observe the animals in a given herd as frequently and regularly as the farmer.

Recall bias is a potential weakness in this retrospective study as the interviews took place approximately one year after the first incursion of pandemic influenza A (2009) virus. Given the Norwegian situation with an outbreak of a previously undiagnosed infectious disease, one would expect farmers to have a heightened awareness and, thus, be more likely to remember and report clinical signs beyond the normal situation. The awareness of pig farmers was also likely affected by the attention given to the outbreak of pandemic influenza A (2009) virus by the public and veterinary health authorities and the media. In addition, the nucleus and multiplier herds in Norway are obliged to keep written records of all treatments of animals, and farmers were encouraged to review these records in a letter enclosed with the questionnaire before the interview. Thus, the high proportion (60%) of positive herds reporting no clinical signs of ILI or increase in reproductive disturbances indicates a high proportion of subclinical infections in cases not complicated by concurrent infections with other respiratory pathogens. The fact that the Norwegian pig population is free from many of the most severe infectious respiratory diseases might lead to a clinical picture less likely to be confounded or masked by concurrent infections. In the present study, only one of the control herds reported clinical signs of ILI.

The low morbidity emphasizes the need for a continued active surveillance program to monitor the status of infection in a naïve pig population. Passive surveillance based on reports of clinical disease would have a low sensitivity as many positive herds would be missed. It also poses challenges when trying to prevent herd-to-herd transmission of pandemic influenza A (2009) virus, as the risks of unintentionally introducing virus-shedding animals to seronegative recipient herds are likely to be increased when the animals are not displaying signs of disease. It also increases the potential risk of pig-to-human transmission. Low morbidity in positive herds indicates a limited economic impact of infection in these herds.

The proportion of infection was approximately the same in both closed, self-replacing nucleus herds and multiplier herds that buy replacement sows from nucleus herds. This supports that introduction of new pigs was unlikely to be the primary source of infection on farms, as previously described by Hofshagen et al..

Information bias is a weakness when open questions are used, especially errors that result from a misunderstanding of questions by respondents. The risk of information bias is, however, reduced in an interview situation by the opportunity to clarify any misunderstandings and by having one person conducting all interviews.

Clinical signs typical of swine influenza were observed in all age groups of animals. Not all infected pigs showed signs even though all were susceptible in the initial phase of infection. Acute outbreaks of swine influenza are more likely to give signs of disease in fully susceptible, seronegative animals. In the present study, the interviews were focused on the alterations in observed signs of disease in the initial phase of the pandemic influenza A (2009) virus outbreak. As described, the Norwegian swine population was free from swine influenza (subtypes H1N1 and H2N3) before the outbreak in 2009. The observation of clinical signs in all age groups of animals might be the case only in the initial phase of infection in previously naïve herds. When a herd has experienced an infection and subsequent seroconversion, later reintroductions or persistence of infections might lead to clinical signs being observed only in age groups of pigs previously unexposed to active or passive immunization against the specific virus. For instance, the morbidity and duration of clinical signs in piglets and weaners could potentially be affected by maternally derived immunity against influenza.

In more than half of the clinically affected herds, decreased feed intake and/or increased reproductive disturbances was reported in sows. These parameters are easily monitored, and farmers use them as reference parameters of performance. As a result, farmers could be more sensitive to alterations in these parameters. None of the control herds reported an increase in reproductive disturbances in sows. The direct role of swine influenza virus in abortions is unclear, and it is commonly believed that the reproductive problems caused by influenza viruses in pigs are due to high fever. Fever was reported in sows, weaners, and growers/finishers/recruit sows, but in this study pyrexia was not emphasized as it was unclear how many farmers routinely checked the rectal temperature of the pigs.

The proportion of observed clinical signs varied between herds and age groups, although the numbers of observations were too small to show statistical significance. This difference could be explained by several factors, the most relevant being herd health status, concurrent infections, differences in sow management, true differences, or recall bias (sows in farrowing unit might “represent” the entire sow population). In addition, some clinical signs (e.g., coughing or sneezing) are more apparent and, therefore, more likely to be recorded and influence the proportion.

In contrast to the low herd morbidity seen in our study, influenza in nonimmune pigs is usually considered to be a disease with high morbidity, low mortality, and with a sudden and remarkable recovery that usually begins within 5–7 days after onset. Experimental infection studies using pandemic influenza (H1N1) 2009 virus have shown a similar clinical picture [8, 9] and reports from natural infections also support this, but with varying morbidity [19, 20]. A recent Australian study in pigs naturally infected with pandemic influenza (H1N1) 2009 virus showed low morbidity and mainly mild clinical signs.

This present study show that nearly 50% (95% CI of 29–77%) of the respondents reported a duration of clinical signs of two weeks or more. The reported duration most likely reflects on presented clinical signs within a herd level or epidemiological unit, so one would expect there to be a prolongation because of pig-to-pig transmission after introduction of virus and incubation time. Reproductive disturbances subsequent to an outbreak of ILI will often be observed and recorded for some time after the acute signs have subsided. This was the case in the present study where 88% of recorded clinical signs in sows lasting two weeks or longer were reproductive disturbances. Concurrent or complicating infections, like bacterial infections, can also prolong the clinical manifestation of respiratory illness.

Zoonotic influenza is a notifiable disease in the Netherlands. Following confirmation of the zoonotic SIV infection, the national and relevant municipal public health authorities were notified and a teleconference was organised to decide on measures. The risk for human-to-human transmission was considered very low, given the enzootic presence of swine influenza viruses and the fact that zoonotic infections are seldom diagnosed.

In order to detect human-to-human transmission at an early stage, it was decided to contact all individuals that had been in close direct contact with the patient without wearing personal protective equipment, and monitor them for symptoms of possible SIV infection (cough, fever or conjunctivitis) for 10 days after exposure. In total, more than 80 contacts were monitored. These included the patient’s family members living in the same household, persons living and working on the pig farm, and healthcare workers who cared for the patient without wearing personal protective equipment (i.e. before the influenza diagnosis). Six contacts developed mild respiratory symptoms including cough, coryza and conjunctivitis during the monitoring period but all tested negative for influenza A virus.

According to the international health regulations, this case has been notified to the European Union Member States and the European Centre for Disease Prevention and Control (ECDC) through the Early Warning and Response System (EWRS) and to the World Health Organization (WHO).

Intranasally delivered LAIV vaccines have been shown experimentally to provide significant cross-protection against heterologous IAV in pigs (7, 8, 32–36). The majority, if not all, of experimental challenge studies in swine with LAIV use pigs that have been procured from high health status herds that are free of many of the bacteria that can be pathogenic under some conditions (i.e., pathobiont) and associated with secondary disease in commercial settings. These bacteria include, but are not limited to, Bordetella, Mycoplasma, Haemophilus and Streptococcus, which are also associated with the respiratory disease complex, a multifactorial disease in which infectious agents, environment, and management practices play a role in susceptibility (37). Much like B. pertussis in humans, B. bronchiseptica in pigs can be isolated from the respiratory tract without evidence of pathology or clinical disease. However, B. bronchiseptica has been shown to cause significant disease when pigs are co-infected with other respiratory viruses, including IAV (17, 18). Inflammation resulting from epithelial and immune cell changes following IAV infection suppresses antibacterial immune mechanisms such that bacterial dynamics and infection severity is impacted [reviewed in Smith and Mccullers (38), Robinson et al. (39)]. LAIV vaccination provides protection against IAV infection, which results in a reduction of secondary bacterial pneumonia (40, 41) and may limit antibiotic usage. However, intranasal administration of LAIV vaccine can alter commensal replication and colonization, though these changes are limited to the upper respiratory tract (23, 42, 43). In the current study, LAIV administration had minimal effect, if any, on B. bronchiseptica density in the upper respiratory tract and we did not appreciate any signs of clinical disease following LAIV administration (data not shown). At day 42 after LAIV administration, which was 49 days after Bb inoculation, there was a significant difference in the amount of Bb in the nasal passages of Bb/NV and Bb/LAIV pigs. However, this could be due to commonly measured shifts in Bb nasal colonization around 7 weeks post-inoculation (25, 26), which is not uncommon. Given that no other changes in Bb colonization following LAIV administration were detected, we do not expect the difference was due to LAIV administration, though it cannot be completely ruled out. It is possible that LAIV administration altered the overall bacterial community structure in the upper respiratory tract of pigs, though additional studies are needed to assess such changes.

The IAV strain used for challenge in this study was not an exact match to the vaccine antigen, as viruses in the β-cluster lineage (based on HA genetic and antigenic characteristics) are distantly related to pdm-lineage viruses (44–46). Thus, as previously shown (8), we anticipated some replication of the challenge virus even in the LAIV vaccinated groups but limited detection in the lower respiratory tract by dpi 5. The presence of lung lesions associated with IAV infection of LAIV vaccinates provided support that IAV replicated in the lower respiratory tract of LAIV/Ch pigs, though there was significant protection against virus replication and pathology. There was not a significant increase in cytokine levels in the lungs of LAIV/Ch pigs compared to control pigs, and levels of IFN-α, IL-1β, and IL-6 in LAIV/Ch pigs were reduced compared to NV/Ch pigs providing support that IAV replication in the lower respiratory tract was readily controlled by LAIV-induced immunity.

Overall B. bronchiseptica colonization did not interfere with LAIV immunogenicity, but LAIV vaccination did not necessarily prevent the negative impact of IAV/B. bronchiseptica co-infection. Although B. bronchiseptica encodes a number of immunomodulatory proteins (47–49), its presence at the time of LAIV administration did not impair induction of mucosal IgA to IAV. We expect LAIV induced the production of mucosal IAV-specific T cells, which has been shown in other species (50); but, the impact of B. bronchiseptica colonization on IAV-specific T cell development is unknown. Although IAV-specific IgA was induced in the upper respiratory tract of pigs following LAIV vaccination during B. bronchiseptica colonization, it was unable to provide full protection upon IAV challenge in the face of B. bronchiseptica infection. Thus, LAIV vaccine based immunity could not protect against the negative interaction that occurred with IAV/B. bronchiseptica co-infection. B. bronchiseptica has a sophisticated environmental sensing system (Bvg locus) that drives expression of numerous virulence factors (51), including unique regulatory system controlling expression in the lower respiratory tract (52). There was minimal pathology in the lungs on Bb-only pigs (Bb/NV/NCh), and lesions present were characteristic of chronic B. bronchiseptica infection, suggesting that virulence gene expression so long after initial B. bronchiseptica inoculation was minimal. However, environmental changes in the respiratory tract associated with IAV infection, such as epithelial damage and/or altered abundance of soluble immune mediators (cytokines, defensins), or leakage of serum proteins into the airway may have subsequently altered B. bronchiseptica gene expression such that cytotoxic factors were produced. Bordetella exposure to albumin enhances production of adenylate cyclase toxin (ACT) (53), and ACT disrupts bronchial epithelial integrity (54). Enhanced expression of B. bronchiseptica virulence factors likely contributed to the appearance of lesions associated with acute B. bronchiseptica infection. The limited replication of challenge virus in LAIV vaccinates also colonized with B. bronchiseptica (Bb/LAIV/Ch) appeared to be enough to incite pulmonary lesions commonly associated with acute B. bronchiseptica infection. Lesion characteristics associated with acute B. bronchiseptica infection were noted in both Bb/NV/Ch and Bb/LAIV/Ch groups, but not the Bb/NV/NCh group. Pathologic changes were not due to an increase in bacterial burden (Figure 4), which has been an ascribed pathologic interaction by other commensal organisms following IAV infection (55), nor significant increases in proinflammatory cytokine levels. Only recently has B. bronchiseptica gene expression in vivo been evaluated (56), and future work aimed at identifying factors that alter gene expression may provide insight on the mechanism by which B. bronchiseptica plays a role in secondary disease.

Mucosal cell-mediated immune (CMI) responses play a role in IAV cross-protection (57, 58), though this was not directly assessed in the current study. B. bronchiseptica may have altered the induction of cell-mediated immunity in the lungs of LAIV vaccinated pigs, though this is unlikely given that IAV-specific IgA was produced and class switching to IgA requires CD4 T cell help. Histologic changes in the lungs of pigs in the LAIV/Ch and Bb/LAIV/Ch group, but not NV/Ch group, consisted of peribronchiolar lymphocyte cuffing and bronchus-associated lymphoid tissue (BALT), indicating that LAIV likely induced a CMI response in the lungs regardless of B. bronchiseptica colonization. Again, changes in the respiratory tract associated with IAV/B. bronchiseptica co-infection, regardless of vaccine status, may hinder immune-protection. It's possible that if the vaccine and challenge antigen were more closely related, such that the LAIV vaccine provided sterilizing immunity against challenge, there would not have been an induction of acute Bordetellosis in the Bb/LAIV/Ch group, though further investigation is necessary to test this hypothesis. Overall, LAIV administration provided significant protection against a distantly related strain of IAV by substantially decreasing replication of the virus in the respiratory tract. Thus, the data presented do not negate the efficacy of LAIV vaccination, but instead indicate that controlling B. bronchiseptica colonization in swine could limit the negative interaction between IAV and Bordetella on swine health.

Given that the LAIV vaccine was delivered by the IN route, the effect of LAIV administration on B. bronchiseptica nasal colonization was assessed at various time points following LAIV vaccination. At the time the initial dose of LAIV was administered, which was 1 week after B. bronchiseptica inoculation, there was no significant difference in B. bronchiseptica colonization between treatment groups (p > 0.05; Figure 1). While there was a numerical trend for differences between groups at days 2 and 3 following the initial dose of LAIV, there was not a statistical difference between groups (p > 0.05). Pigs in the LAIV group were boosted at day 21, and again, there was not a significant difference in B. bronchiseptica colonization between treatment groups at the time of LAIV administration (day 21), nor 1 week following administration of the booster dose (day 28, p > 0.05). Only on day 42 relative to LAIV administration was there a significant difference (p < 0.05) in B. bronchiseptica CFUs in nasal wash between Bb/LAIV and Bb/NV treatment groups, though on average, it was limited to less than a log10 difference (4.72 ± 0.92 vs. 3.89 ± 0.43, log10 CFU ± SEM/ml respectively).

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.

All the inoculated pigs showed typical clinical symptoms, namely, sneezing and coughing. They also developed a high fever, and their average rectal temperature exceeded 40.0°C between PID 2 and 4. In contrast, control pigs exhibited no clinical symptoms throughout the experiment. The three groups infected with H1N1, H1N2, and H3N2-infected groups did not differ significantly in their clinical appearance that included sneezing, coughing, rectal temperature, and respiratory rate (data not shown).

The viral RNA of H1N1 was detected in lung samples from the infected pigs at PID 2 (2/3; number of positive pigs/number of pigs tested), PID 4 (3/3), PID 7 (2/3), and PID 14 (1/3), while the viral RNA was detected in the pigs infected with H1N2 or H3N2 at PID 2 (1/3 or 3/3), PID 4 (3/3 or 3/3), and PID 7 (2/3 or 2/3), respectively. However, pigs of the control group remained negative for the viral RNA throughout the study period.

The gross lung lesions of infected pigs ranged from multifocal lesions to a coalescing reddish-tan consolidation of the lung, particularly affected the cranial lobes, but these lesions were not detected in the control pigs (Figure 1). The three infected groups differed in their gross lesion scores following post inoculation. The H3N2 infected pigs showed significantly higher lung lesion score than other groups (P < 0.05) at PID 2, and this then progressively decreased. Both the H1N1 and H1N2 infected pigs lacked gross lesions at PID 2, but they showed minimal to mild pneumonia lesions on PID 4, 7 and 14. The pigs infected with H1N1 had significant scores (P < 0.05) of gross lesions when compared with the other pigs infected with H1N2, H3N2, and control at PID 14 (Figure 2A.).

The histological lesions were limited to the bronchi, bronchioles, and alveoli, but no lesions were observed in the tissues of control pigs. The pigs infected with H1N1, H1N2, and H3N2 all shared the following histopathological features; bronchial/bronchiolar necrosis, thickening of the alveolar septa due to the infiltration of inflammatory leukocytes, and pulmonary hemorrhage and atelectasis (Figure 3). The H3N2 infected pigs had severe histological lesions at PID 2, while, in the other sub-type infected pigs, the lesions were milder at PID 2. However, at PID 14, these lung lesions of H1N1-infected pigs had a significantly higher mean histological score (5.3, P < 0.05) than those from the lungs of H1N2 (0) and H3N2 (2.1) infected pigs (Figure 2B).

SIV was detected in the lung tissue following IHC staining. Influenza viral antigens were predominantly found in the bronchial and bronchiolar epithelium. In addition, numerous necrotic epithelial cells that underwent phagocytosis by alveolar macrophages were detected in the lumen. In the three infected groups, mean antigen-positive score of the viral antigens of H3N2-infected pigs was higher than those of the other group’s pigs at PID 2. The mean antigen scores of pigs infected with H1N2 and H3N2 were rarely detected from PID 7. However, an increased amount of viral antigens in H1N1infected pigs were found in the bronchioles and alveolar epithelium, and the mean score was significantly higher (P < 0.05) than those of H1N2 and H3N2 infected pigs at PID 14 (Figures 4 and 5).

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.

Swine influenza is commonly characterized by fever, respiratory and systemic nonspecific symptoms (i.e. loss of appetite, apatia). In the enzootic form, clinical signs may be less obvious. Subclinical infection is also quite common [17, 21, 22]. During uncomplicated infection the morbidity can be as high as 100% but the mortality is relatively low (ranges from less than 1% to 4%). The most common complications of swine influenza are secondary bacterial pneumonia and PRDC [3, 12]. Coinfections often lead to overproduction of cytokines that may be harmful to the host. There are several mechanisms, by which SIV infection predisposes to secondary bacterial infection, including: increased expression of cell receptors leading to increased colonization and modification of host immune responses (i.e. impairing of phagocytic function of alveolar macrophages) [23–26]. Furthermore, it has been shown that damage caused by SIV in the respiratory tract (i.e. loss of cilia, extrusion of mucus, exudation, necrosis and metaplasia of airway epithelium), can reduce the ability of the host to clear the bacterial superinfection [2, 21]. Hps is one of the pathogens which may complicate swine influenza and be one of the etiological agents of PRDC [2, 3, 8, 27].

The mechanism by which SIV affects the host’s susceptibility as well as its immune response to secondary bacterial infections has not been fully elucidated. Previous research reviled that interactions among multiple pathogens can lead to an exacerbated inflammatory response and increased severity of infections [12, 28–30]. For example, gross lesions in the lungs and magnitude of APP response in pigs co-infected with SIV and Pasteurella multocida were more intensive compared to animals infected only with SIV or Pasteurella multocida. Similar results were observed in pigs co-inoculated with SIV and A. pleuropneumoniae. Co-infection with SIV and A. pleuropneumoniae potentiated the severity of lung lesions caused by SIV and enhanced virus replication in the lung and nasal SIV shedding. Enhanced SIV replication contributed to a more severe clinical course of the disease as well as earlier and higher magnitude immune and inflammatory responses. Loving et al. reported that SIV infection increased Bordetella bronchiseptica (Bb) colonization and increased the production of proinflammatory cytokines likely to exacerbate lung lesions. Pulmonary lesions in the co-infected pigs were more intense compared to SIV-only or Bb-only groups. The type I interferon, IL-1β and IL-8 were also significantly elevated in lungs of co-infected pigs.

There is limited data on the influence of SIV and Hps coinfection on the clinical course, kinetics of the immune and inflammatory response, as well as pathogen load and shedding. Both microorganisms are frequently isolated from respiratory tract of pigs in the field conditions [1, 27]. A previous study, investigating the role of prior SIV infection on the Hps colonization, revealed that Hps colonization was higher in the nose and lungs of SIV/Hps pigs compared to Hps-only pigs. These results indicate that SIV infection contributes to enhance bacterial colonization. In SIV/Hps pigs IL-8, IL-6 and IL-1β protein levels were increased in the tracheal wash and bronchoalveolar lavage fluid (BALF), and BALF cells IL-8, IL-6 and IL-1β mRNA expression levels were significantly increased over SIV-only and Hps-only pigs.

Based on the results of our study it could be stated that Hps did not cause significant lesions in the lung unless pigs were co-infected with SIV. No significant macroscopic lung lesions after experimental infection with Hps has been also reported previously. In the same study Hps was not isolated from Hps-single infected pigs. In our experiment we had been able to isolate of Hps in the case of 3 out of 11 Hps-single inoculated pigs, but in group co-inoculated with SIV the isolation was successful in 8 out of 11 animals. In the remaining groups (SIV and control) Hps was not isolated from lung. Typical systemic lesions of polyserositis were found in only one pig from co-inoculated group. In the present study no significant differences between gross lung lesions has been found between groups single or dually inoculated with SIV but in both groups lung score was significantly higher than in Hps-single inoculated group. In addition, more severe clinical signs were observed in dual inoculated pigs compared to single Hps- but not SIV-inoculated animals. The more severe clinical course of the infection was probably a consequence of more severe lung lesions present in pigs inoculated with SIV or SIV and Hps. Enhanced lung lesions in pigs single or dual inoculated with SIV could be a result of stronger replication of infective agents and more severe local inflammatory responses. Similarly as in the previous study establishing that the titre of Bb was higher in the respiratory tract of SIV/Bb co-infected pigs, we found that SIV enhanced Hps colonization of the lung. In accordance to other experiment the significantly higher SIV titre in the nasal swabs and lung was observed in co-inoculated pigs. Simultaneously, no effect of SIV on the Hps shedding has been found. These findings suggest that Hps can facilitate SIV replication in the respiratory tissue of dual inoculated pigs.

The significant influence of both pathogens on the systemic inflammatory response has been also found in the present study. In the groups inoculated with bacteria (single or dual) the APP response was in general higher than in virus-single inoculated animals. These findings are in accordance with the results of previous studies with various respiratory pathogens of swine [12, 19, 31, 32].

Depending upon the involvement of etiological agent, the infectious respiratory diseases of small ruminants can be categorized as follows [9, 14]:bacterial: Pasteurellosis, Ovine progressive pneumonia, mycoplasmosis, enzootic pneumonia, and caseous lymphadenitis,viral: PPR, parainfluenza, caprine arthritis encephalitis virus, and bluetongue,fungal: fungal pneumonia,parasitic: nasal myiasis and verminous pneumonia,others: enzootic nasal tumors and ovine pulmonary adenomatosis (Jaagsiekte).

Manytimes due to environmental stress, immunosuppression, and deficient managemental practices, secondary invaders more severely affect the diseased individuals; moreover, mixed infections with multiple aetiology are also common phenomena [5, 8, 13, 15].

These conditions involve respiratory tract as primary target and lesions remain confined to either upper or lower respiratory tract [7, 16]. Thus, these diseases can be grouped as follows [5, 8, 14, 17].Diseases of upper respiratory tract, namely, nasal myiasis and enzootic nasal tumors, mainly remain confined to sinus, nostrils, and nasal cavity. Various tumors like nasal polyps (adenopapillomas), squamous cell carcinomas, adenocarcinomas, lymphosarcomas, and adenomas are common in upper respiratory tracts of sheep and goats. However, the incidence rate is very low and only sporadic cases are reported.Diseases of lower respiratory tract, namely, PPR, parainfluenza, Pasteurellosis, Ovine progressive pneumonia, mycoplasmosis, caprine arthritis encephalitis virus, caseous lymphadenitis, verminous pneumonia, and many others which involve lungs and lesions, are observed in alveoli and bronchioles.

Depending upon the severity of the diseases and physical status of the infected animals, high morbidity and mortality can be recorded in animals of all age groups. These diseases alone or in combination with other associated conditions may have acute or chronic onset and are a significant cause of losses to the sheep industry [3, 10]. Thus, the respiratory diseases can also be classified on the basis of onset and duration of disease as mentioned below [3, 9, 14, 18]:acute: bluetongue, PPR, Pasteurellosis, and parainfluenza,chronic: mycoplasmosis, verminous pneumonia, nasal myiasis, and enzootic nasal tumors,progressive: Ovine progressive pneumonia, caprine arthritis encephalitis virus, caseous lymphadenitis, and pulmonary adenomatosis.

In the present study, we have shown that H3N2 SIV cause massive damage to the bronchial and bronchiolar epithelial cells at the start of infection (PID 2) eliciting gross and histological lesions. The inflammation caused by H3N2 may affect the initial stages of infection. In comparison, H1N1 or H1N2 infected pigs exhibited few (histological) or no (gross) pathological changes at this stage. These results are linked to the mean antigen-positive scores from infected pigs, which demonstrated that the viral antigens were not as widely distributed in the lungs of pigs infected with H1N1 or H1N2 at PID 2 when compared to H3N2 infected pigs. Although the H1N1detection in a pig’s lung of three pigs at PID 14 might be caused by second infection between pigs within the same group, no virus was observed by IHC and RT-PCR in other pigs infected with H1N2 or H3N2.

It is acknowledged that bronchial and bronchiolar epithelial cells and leucocytes found in inflammatory lesions are the main source of pro-inflammatory cytokines [14, 15]. Thus, tissue damage may be responsible for the severe suppurative, necrotizing bronchitis and broncho-interstitial pneumonia observed at the beginning of infection (PID 2) in H3N2-infected pigs.

Subsequently, between PID 4 and 7, inflammation with lymphocytic infiltration and epithelial proliferation were found in the lung tissues from pigs infected with H1N1, H1N2, and H3N2. Influenza viral antigens were also frequently found in the alveolar macrophages within the thickened alveolar septa and the peribronchiolar lymphatic vessels. The gross and histological pulmonary lesions from pigs infected by H1N2 or H3N2 gradually decreased between PID 4 and PID 14, and the mean antigen-positive scores decreased in the lungs from both H1N2 and H3N2 at PID 7. However, the pigs infected with H1N1 SIV showed significantly high levels of the viral antigen distribution, as well as increased gross and histopathological lung scores.

Based on these data, we have demonstrated that H1N1 SIV, isolated from Korea, may replicate in the lung and can induce pneumonic lesions for a relatively longer period than the other two viruses; H1N2 and H3N2. This comparative investigation of SIV isolates from Korea is the first study of their pathology, since the pathogenesis of SIV has only been studied in pigs experimentally infected by each SIV subtype in Korea [3, 16, 17].

Here, our pathological study demonstrated that Korean SIV subtypes have different pulmonary pathologic patterns. The H3N2 rapidly induces acute lung lesions, such as broncho-interstitial pneumonia, while the H1N1 is associated with pulmonary lesions later in infection. This may reflect differences in the speed of initial replication rates in bronchial and bronchiolar epithelial cells. These observations also demonstrate the importance of comparing the pathogenesis of influenza viruses over the entire course of an infection as opposed to an individual time-point.

Many groups believe that PCV2 alone is not enough to induce PCVD/PCVAD. However, PCV2 targets lymphoid tissues and strongly impacts T-cell selection processes in the thymus, resulting in an obvious lymphoid depletion and immunosuppression in the pig. Consequently, PCV2 infection results in an increased susceptibility to opportunistic infections of viruses and bacteria. Obviously, co-infections of PCV2 with other viruses may increase pathogenicity in pigs, resulting in more severe clinical symptoms. In addition to dual infections with PCV2 and other viruses, multiple infections are often detected in the field. Moreover, many lines of evidence have shown that bacterial infection, vaccination failure, stress or crowding together with PCV2 can also lead to PCVD/PCVAD. In addition, some PCV2-infected pigs can develop severe diseases; however, PCV2 also evokes a subclinical infection in pigs without any obvious symptoms in many cases. Therefore, PCV2 increases clinical signs of PRRSV, CSFV, SwIV, PRV or PEDV infections, and meanwhile, secondary infection (such as PPV infection) and worse physical/growth condition can also provide a better in vivo environment for PCV2 infection. Thus, more research is needed to improve our understanding of the interactions between different swine viruses and bacteria during co-infection with PCV2 in pigs, including how they interact with the host immune response and how they affect the efficacy of vaccination. These studies could lead to important breakthroughs in the understanding of PCVD/PCVAD and in the development of new strategies to control the disease.

In conclusion, we found evidence of zoonotic transmission of MERS-CoV in Morocco, supporting previous data from Kenya and indicating that MERS-CoV is capable of zoonotic infection in Africa. However, it is not yet known if these MERS-CoV circulating in Africa can cause zoonotic disease, i.e. invade the lower respiratory tract to cause severe viral pneumonia. The genetically diverse strains of MERS-CoV found in Moroccan dromedaries, as well as other regions of western Africa, e.g. Nigeria and Burkina Faso, might have a lower potential for severe zoonotic disease compared with the virus strain isolated in the Middle East. Testing for MERS-CoV RNA in patients with severe acute respiratory infections in dromedary camel herding regions of Africa is needed to ascertain whether or not zoonotic MERS disease is occurring in Africa.