Haemophilus parasuis: Infection, immunity and antibiotics

H. parasuis is considered one of the most important bacterial pathogens affecting pigs. Vaccines and other management strategies have not always been successful in controlling the losses associated to H. parasuis. This bacterium frequently colonizes the mucosal of the swine upper respiratory tract. Oliveira et al., (2004) showed that exposure of young pigs to a low dose of virulent H. parasuis strain (controlled exposure) reduced mortality due to Glasser's disease and could induce the development of protective immunity. Under field conditions, factors that may disrupt colonization patterns of H. parasuis at weaning, such as antibiotic treatment, might result in delay of disease to late nursery phases, due to lack of immune response priming.

H. parasuis, as a gram-negative bacterium, interacts with the immune system at different levels, including the innate and the specific immune system. The literature offers a vast characterization of antibody-mediated immune responses, whereas little information is currently available for innate immune and cell-mediated responses. A recent study showed that H. parasuis susceptibility to phagocytosis by porcine alveolar macrophages (PAMs) correlates with the clinical origin of the strain. H. parasuis strains isolated from systemic lesions (virulent) were resistant to phagocytosis, while nasal strains (non-virulent) were efficiently phagocytosed by PAMs in vitro, followed by subsequent bacterial death within the macrophage. Similarly, H. parasuis was able to stimulate the production of IL-1 expression in lung of pigs undergoing severe disease following experimental infection, whereas IL-4, IL-10, tumor necrosis factor-alpha (TNF-α), and (IFN-γ) were expressed in significantly higher levels in spleen, pharyngeal lymph nodes, lung and brain of survivors, which suggests that these cytokines might contribute to protection against H. parasuis. Specific increases in the relative proportions of T and B lymphocytes isolated from peripheral blood mononuclear cells (PBMC) were also found in colostrum-deprived pigs after challenge with virulent H. parasuis strain, but not after immunizations.

Most frequently, pigs exposed to H. parasuis live cultures or vaccinated with killed bacterins generate progressively increasing serum IgG antibody response. Pigs with high homologous titers are protected against challenge, while Glasser's disease has been associated with absence or low titers of serum antibodies. Likewise, pigs lacking maternal immunity were susceptible to Glasser's disease upon inoculation with a virulent H. parasuis strain, whereas pigs that received maternal immunity were protected. Cerda-Cuellar et al. (2010) further demonstrated that piglets from vaccinated sows had significantly higher levels of antibodies earlier after birth and were colonized later and to a lower degree than piglets from non-vaccinated sows. Furthermore, the increase in colonization rate was associated with a decrease in H. parasuis serum antibodies in piglets, which indicates that the level of maternal antibodies in piglets might be able to modulate the timing and level of colonization by H. parasuis.

According to Oliveira et al., (2004), the success of H. parasuis controlled exposure in preventing mortality during the nursery stage suggests that H. parasuis might interact with the mucosal of the upper respiratory tract, possibly resulting in colonization, priming of the immune response and protection from disease. Therefore, the development of a protective immune response against H. parasuis might be delayed by factors that interfere with bacterial colonization. Recent studies have shown that early antibiotic treatment not only prevented the development of an effective immune response against Chlamydia trachomatis and Salmonella sp., but also rendered the animals susceptible to challenge infection. In contrast, however, a recent study demonstrated that early antibiotic treatment with enrofloxacin against Salmonella typhimurium infection primed specific antibody response, which protected against secondary challenge. Enrofloxacin, the primary compound of Baytril (Bayer Animal Health), is considered efficacious against H. parasuis, but limited information is currently available on the effects of enrofloxacin on H. parasuis colonization and immunity. Therefore, a better understanding of H. parasuis colonization over time and the effect of antibiotics on colonization and the development of an effective immune response requires further investigation in order to prevent and control disease.

The objective of our study was to evaluate the effect of enrofloxacin (Baytril 100) in H. parasuis colonization in weaned pigs. Additionally, in order to recreate H. parasuis colonization under conditions representative of what occurs in the field, a new experimental inoculation model was developed in conventional pigs to better understand dynamics of colonization and immune responses to virulent H. parasuis.

 

Forty five pigs were identified in a commercial herd and screened for H. parasuis using gel-based PCR.15 Twentyfour of the weaned pigs that tested positive for H. parasuis were selected and moved to the University of Minnesota research isolation facility. Pigs were randomly divided into one treatment group of 12 pigs and one control group of 11 pigs and housed in two separated rooms (one pig in the control group died shortly after arrival). On arrival at the research facility, blood samples and nasal and tonsil swabs were collected from all the pigs. Pigs in the treatment group were treated with a single dose of injectable enrofloxacin (0.034ml/lb / 7.5 mg/Kg Baytril) at 24 h post arrival. Pigs in the control group received saline solution intramuscularly but not antibiotics. Pigs were monitored for 15 days. Throughout the study pigs were sampled by tonsillar and nasal swabs and selected pigs were necropsied at 3, 8 and 14 days post-treatment (DPT). At necropsy, swabs from the nasal cavity, tonsil, trachea, lung, and peritoneal and pleural serosas were collected for diagnostic investigation. Nasal swabs were processed for qPCR testing. DNA from swabs were extracted using DNeasy Blood & Tissue Kit Qiagen kit and then tested individually by quantitative PCR (qPCR),16 with some modifications. Differences between the proportions of H. parasuis positive pigs in treated vs control groups at each sampling time point were calculated using Fisher’s Exact Probability Test. The Bonferroni correction was used to address multiple comparisons (α = 0.003).

Sixteen conventional weaned pigs were divided into 3 groups and placed into 3 different rooms at the University of Minnesota research isolation facility. At day 0 of the study, groups 1 and 2 (n = 6 each) and 3 (n = 4) received 10⁶ or 10⁴ CFU/ml of highly virulent H. parasuis, strain Nagasaki, or saline, intranasally, respectively. Clinical evaluation and nasal swabs (for bacterial culture) were collected before and every day after inoculation (dpi) during 7 days. Blood samples were collected on 1, 3 and 4 dpi for bacterial isolation. At 2 time points (4 and 7 dpi), half of the pigs were euthanized and assayed for the presence of H. parasuis in the respiratory tract and systemic sites. H. parasuis isolates obtained were genotyped by ERICPCR in order to differentiate strain Nagasaki from any other H. parasuis strains that the pigs may carry.

 

Pigs tested positive by gel-based PCR at the herd of origin and on the day of arrival. Results from qPCR showed that twenty two out of twenty three pigs (95.7%) tested positive from tonsil swabs on arrival, while only 11 pigs (48%) tested positive from nasal swabs. All treated pigs tested H. parasuis negative by qPCR at 1 day post-treatment (DPT). Moreover, treatment effect persisted partially until 12 DPT. The control pigs tested positive throughout most of the days of the study. Differences between the proportion of positives for control and treated pigs were statistically significant on days 1, 2, 3, 4, 5, 6 and 7 post-treatment for nasal swabs and on days 2, 4 and 5 post-treatment for tonsil swabs (P-value < 0.003). At necropsy, nine out of eleven control pigs were positive for H. parasuis in at least one of the five samples tested. One sample from the serosas was cultured positive and two isolates were recovered from the nasal cavity. In contrast, only 4 out of 12 pigs in the treatment group tested positive at necropsy. Three pigs tested positive in the tonsil, 3 in the trachea, and one in the nasal cavity. None of the serosas were cultured positive and only one bacterial isolate could be cultured from the nasal cavity at necropsy. Interestingly, all isolates were recovered at 15 DPT. No clinical signs or lesions of any kind were observed during the experiment.

ERIC-PCR genotyping demonstrated that the H. parasuis strains isolated before inoculation were identical, and frequently isolated from the nose of all the pigs throughout the study. The Nagasaki strain, also identified by ERIC-PCR, was recovered from the nose of 5 pigs after inoculation. Moreover, tracheal swabs collected at necropsy yielded only Nagasaki isolates in 10 pigs. Overall the Nagasaki strain was isolated at least once from all 12 inoculated pigs, but was never recovered from systemic sites of experimentally inoculated pigs, control pigs or before inoculation. There were no differences in isolation of the Nagasaki strain based on inoculation doses. The absence of fever, clinical signs, lesions and bacteremia demonstrates that there was no systemic infection, even though the Nagasaki strain can be highly virulent. Trachea represents a less competitive niche for H. parasuis colonization, which may explain why tracheal swabs yielded higher number of Nagasaki isolates compared to nasal swabs.

 

Under the conditions of this study, enrofloxacin treatment significantly reduced the number of pigs colonized with H. parasuis and this effect was mostly seen during the first week post treatment. In addition, enrofloxacin also reduced the presence of H. parasuis on the nasal cavity and the tonsils of naturally colonized pigs, but was unable to completely eliminate the organism. Nevertheless, the results showed that the effect of enrofloxacin was rapid and active for relatively long time against H. parasuis harbored on the nasal cavity and tonsils of weaned pigs, since all treated pigs tested negative at 1 DPT and continued to test negative during most of the study.

Reduction of H. parasuis in the nasal cavity and tonsils may help to control the disease during susceptible stages such as the weaning period, but further research is needed to evaluate the lasting effect of enrofloxacin in colonization patterns and immune response. We intend to investigate these findings further by using the H. parasuis colonization model that we have established in conventional pigs to study the early events of H. parasuis infection and immune response in conventional pigs.

 

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