Introduction

Respiratory infectious diseases are a limiting factor for camelid production in the Peruvian high Andes because they cause high morbidity and mortality rates, especially during the first months of life, and Pasteurela multocida is commonly isolated from camelids with respiratory disease (Rosadio et al. 2011).

P. multocida is a normal member of upper respiratory tract microbiota in a wide variety of species. However, stress caused by environmental factors (extreme cold), viral infections, and immunosuppression promote bacterial invasion of lung tissue and development of pneumonia (Hodgson et al. 2005). P. multocida is responsible for a wide range of infections in both domestic and wild animals, causing bronchopneumonia and hemorrhagic septicemia in bovines, atrophic rhinitis in swine, fowl cholera in birds, and human infections following animal bites (Quinn et al. 2015).

P. multocida has several virulence factors that included capsule, lipopolysaccharide (LPS), the P. multocida toxin (PMT, a member of dermonecrotic toxin family), and iron-regulated and iron-acquisition proteins, in addition to other virulence factors, which play an important role in the pathogenesis, and these may differ among infections and hosts (Ewers et al. 2006).

In this study, we investigated P. multocida isolates from an outbreak of pneumonia in young alpacas using specific PCR reactions to identify capsule and LPS type and key virulence factors. Furthermore, enterobacterial repetitive intergenic consensus (ERIC) PCR-based (ERIC-PCR) was used to identify and determine genetic diversity among the P. multocida strains.

Material and methods

Samples and P. multocida isolation

Lung tissues were obtained from 46 dead alpacas (Vicugna pacos) between 1 and 2 months of age, from three distinct herds, located in the Experimental Center “La Raya,” in the Department of Puno, during the months of January to February of 2014. At necropsy, all animals had lesions compatible with pneumonic disease, and the time between death of the animal and sampling was within a maximum of 2 h.

Lung tissue fragments were immediately used to inoculate triptic soy agar (Mast Group Ltd., Merseyside, UK) supplemented with 5% sheep blood plates. Plates were then incubated at 37 °C for 24 h under aerobic conditions. Isolates with positive reaction to catalase, oxidase, and indole production tests were analyzed by API 20NE (Biomerieux, France) biochemical identification kit, performed following the supplier recommendations, for initially identify those isolates belonging to the P. multocida species. Suspected colonies of P. multocida were inoculated in vials containing 2 ml brain heart infusion broth (Mast Group Ltd., Merseyside, UK) and were grown for 24 h at 37 °C for genomic DNA extraction, which was performed using the Purification Wizard Genomic DNA Kit (Promega, Madison, WI, USA) according to manufacturer’s instructions.

PCR for virulence genes detection

A primer pair specific for the PCR amplification of a unique P. multocida region in the kmt1 gene was used as described by Townsend et al. (1998). PCR reactions used to identify the 5 capsular types and 16 LPS genotypes were performed using primers described by Townsend et al. (2001) and Harper et al. (2015), respectively.

PCR reactions specific for the detection of the virulence associated genes toxA (P. multocida toxin, or PMT), tbpA (transferrin binding protein A), hgbB (hemoglobin binding protein), and pfhA (filamentous protein), were carried out with primers as described by Ewers et al. (2006). Positive controls for each analyzed gene were donated by the Universidad Complutense de Madrid, Spain (P. multocida A and D from ovine source).

ERIC-PCR

ERIC-PCR analysis, using primers targeting palindromic sequences, according to Versalovic et al. (1991) was done and the observed bands in the gel were evaluated based on the presence or absence of polymorphic fragments by ERIC primers. Cluster analysis dendrogram was performed with NTSYSpc V. 2.1, based on Dice’s similarity coefficient (SD) and the unweighted pair group method with arithmetic mean (UPGMA). A similarity genetic index of ≥88 was used to define clusters among isolates.

Results

P. multocida was recovered from 52% (24/46) samples of tissue lung from mortal pneumonia cases in young alpacas. All isolates with positive reactions for catalase, oxidase, and indole production were confirmed as P. multocida by API20NE and amplification of the kmt1 gene fragment by PCR. PCR reaction for capsular and LPS typing and genes encoding virulence factors reveals that all isolates belonged to capsular type A (presence of hyaD-hyaC gene) and L6 LPS genotype (confirmed by the presence of nctB gene, corresponding to the Heddleston serotypes 10, 11, 12, and 15) and possessed the toxA and tbpA gene, which encoding for P. multocida toxin (PMT) and transferring protein binding A, respectively. The ERIC-PCR electrophoretic pattern among P. multocida isolates showed two groups, where almost all isolates (23/24), were grouped in a single cluster (Fig. 1), revealing clonal spread among individuals of a single strain of P. multocida.

Fig. 1
figure 1

A dendrogram representing the genetic relationship between all P. multocida isolates based on the presence/absence of ERIC-PCR fragments. The dendrogram was built using the Unweighted Pair Group Method with Arithmetic mean (UPGMA) using the Numerical Taxonomy and Multivariate Analysis System (NTSYSpc v. 2.1) program based on Dice’s similarity coefficient (SD). Clusters were defined based on arbitrary cut-off value of 88% genetic similarity. Strains analyzed in this study are showed on the right with capsule and LPS typing and virulence factors encoding genes. A single asterisk indicates control strains

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Discussion

In this study, we described the first genetic characterization of P. multocida from pneumonia cases in alpacas. Alpaca pneumonia is related with high mortality rates in young animals, and the main agents associated with acute respiratory diseases, in addition to P. multocida, is Manheimia haemolytica, described previously (Rosadio et al. 2011); however, in this work, M. haemolytica was not isolated. M. haemolytica was described by Rosadio et al. (2011) as causing severe lung injury in neonate alpacas.

P. multocida type A strains affect a wide range of host species (Quinn et al. 2015), and has been reported previously as primary cause of respiratory disease in a wide range of ruminants (Kumar et al. 2009). P. multocida toxA+ is principally associated with atrophic rhinitis lesions in pigs and rabbits (DiGiacomo et al. 1989; Ewers et al. 2006) and pneumonia in birds and sheep (Ewers et al. 2006; Shayegh et al. 2008). ToxA gene, that encoding for P. multocida toxin (PMT), plays an important role in the pathogenesis of atrophic rhinitis, and mediated their pathogenic effect in the cells by disruption of multiple signaling pathways (Wilson and Ho 2011). This gene is mainly detected in P. multocida type D, and less frequently in Type A strains (Ewers et al. 2006). TbpA gene, encoding for an iron acquisition protein, is strongly associated to ruminant strains causing pneumonia and hemorrhagic septicemia (Ewers et al. 2006).

In this work, we use a LPS genotyping protocol described by Harper et al. (2015), instead of classical Heddleston LPS serotyping, due to time consuming, no access to specific antisera and lack of accuracy and reproducibility of this technique. This is based on the fact of that 16 LPS types produced by P. multocida are generated by 8 distinct loci, named L1 to L8, and variation in the structure due to random point mutations or deletions on the LPS sequences displayed all the Heddleston LPS types (Harper et al. 2015). With this protocol, we report that P. multocida isolates belonged to the L6 LPS genotype. This genotype is also found in P. multocida strains isolated in various species of domestic birds (chicken, turkey, and ducks) and mammals (pigs). The knowledge about P. multocida LPS genotypes allows a better understanding about the epidemiology of the infection (Harper et al. 2015), but apparently, for the vaccine development, this information is not essential, at least this was observed in L1 and L3 LPS genotypes, where a vaccine containing a LPS specific type confers protection against others LPS types (Harper et al. 2016).

ERIC-PCR typing of the 24 isolates, found that 96% (23/24) of the isolates displayed a unique profile, showing a close relationship among the majority of isolates, suggesting that a single virulent strain of P. multocida caused the pneumonic infections by clonal spread among susceptible hosts in the herd. Outbreaks of infectious pneumonia in young alpacas are very common, especially in the months of intense rains (January and February), and in alpaca shearing due to the stress caused in the animal handling.

The present study is the first genetic characterization of P. multocida isolated from high Andean alpacas with pneumonia. This information presented contributes to knowledge of the etiology and pathogenesis of the disease, provides a basis for the development of control and prevention strategies, and documents the importance of molecular typing techniques for the determination of genetic variability among the P. multocida isolates.