Biological proprieties of Pasteurella multocida isolate from sheep in Morocco

Background In Morocco, observations of pasteurellosis clinical cases reported by �eld veterinarian and lesions observed in abattoirs, suggest circulation of serotypes of pasteurellosis among small ruminants population. However, little is known on the prevalence of the main serotypes in this region and their pathogenicity. Results We investigated �ve suspected Pasteurella clinical cases from different provinces of Morocco. We succeeded to isolate 2 strains (S14 and S13) of Pasteurella Multocida A among the �ve isolates. Identi�cation was achieved by biochemical and molecular biology methods. Phylogeny based on two genes sequence analysis (RNA16S and rpoB) suggested that the two isolates present similarity with others from different species. A pathogenicity study was conducted in mice, guinea pigs and sheep to set up a model for vaccine testing. The strain S14 was more virulent than S13 in laboratory animals and induced severe illness in sheep. Conclusion The high mortality of infected mice suggest that this animal may represent a good alternative for testing pathogenicity and vaccine e�cacy.


Introduction
Morocco is a north African country with a large small ruminant population, 24 million, that occupy different biotopes.This population is relatively well supervised by experimented veterinary staff, belonging to private or public sector.Vaccination campaigns are regularly conducted against major small ruminant diseases such as Peste des petits ruminants, Sheep pox, Goat Pox and clostridia.However, observations of pasteurellosis clinical cases reported by eld veterinarian and lesions observed in abattoirs, suggest circulation of different serotypes of pasteurellosis among small ruminants population in Morocco.Pasteurellosis in small ruminants has never been reported in the country and no scienti c study was published on the identi cation and/or isolation of Pasteurella and especially there are almost no reports on the pathogenicity of Pasteurella originated from small ruminants.
Pasteurellosis is caused by Pasteurella multocida, Pasteurella haemolytica (reclassi ed in 1999 as Mannheimia haemolytica) and Pasteurella trehalosi.Pasteurella and Mannheimia organisms are gramnegative, non motile, coccobacillary, non-sporing, fermentative and facultative anaerobe of the Pasteurellaceae family.This family tends to inhabit the mucosal surfaces of the gastro intestinal, respiratory and genital tract of mammals.Pasteurella serotypes have different levels of virulence, hostspecies adaptability with possible inter-species transmissibility, antigenicity, immunogenicity, drug resistance and a lack of inter-serotype cross-reactivity [1,2].
M. haemolytica and B trehalosi are distributed worldwide, and diseases caused by them are common in sheep and goats of all ages, although the prevalence of serotypes may vary by region and ock.M haemolytica is the most commonly isolated bacteria in clinical cases, followed closely by P trehalosi, P multocida is seen less frequently.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 bacteria invasion of lung tissue and development of pneumonia.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 [3][4][5][6].Infections with P. multocida lead to great economic losses in the farming industry due to their severe morbidity and mortality.The species multocida is divided into three subspecies, namely P. multocida subsp.gallicida, P. multocida subsp.multocida and P. multocida subsp.septica, mainly on the basis of the ability to ferment dulcitol and sorbitol [7].P. multocida is classi ed into ve capsular serogroups (A, B, D, E and F) on the basis of capsular antigens and into 16 somatic serotypes, based on lipopolysaccharide and likely to be associated with speci c types of disease [6,8].P. multocida serotypes B and E cause haemorrhagic septicaemia of cattle, buffalo, goat, camel and deer; serotype D causes atrophic rhinitis of pigs and rabbits; serotypes A and D cause enzootic pneumonia and shipping fever of cattle, sheep and pigs; and serotypes A and F cause avian cholera of all bird species [9][10][11].Besides poultry, P. multocida in this serogroup has also been isolated from rabbits [12].
In this study, we investigated ve suspected Pasteurella clinical cases in different regions of Morocco.We studied biological characteristic and pathogenesis in laboratory animals.We conducted molecular sequencing of the isolates and a challenge study on sheep and mice to set up a model for vaccine testing.The resulting pathogenesis in sheep was evaluated by following disease symptoms, lesions and bacterial charge in different tissues.This is the rst time a Pasteurella strain was isolated in the small ruminant population of Morocco and characterized by sequencing and pathogenicity in sheep and laboratory animals.

Culture and biochemical identi cation
Lung tissue fragments were used to inoculate triptic soy agar supplemented with 5% sheep blood plates.
Plates were then incubated at 37°C for 24 h under aerobic conditions.Colonies giving gram-negative coccobacilli or short rods with or without bipolar staining on smears were subcultured for identi cation.A 24-hour pure Pasteurella suspected culture was also subjected to biochemical tests (catalase, oxydase and indole) and analysed by API 20NE (Biomerieux, France) biochemical identi cation kit, performed according to the supplier instructions.

Inoculum preparation
The organism was seeded in BHI (Brain Heart Infusion) broth and incubated at 37°C with shaking at 100rpm.The log phase cultures, used for injection animals, were prepared in BHI broth.The optical density (OD) of the culture was measured using 600nm wavelength light source.Bacterial concentration was determined by quanti cation via serial dilution.
The ampli ed product was separated by electrophoresis on 1% agarose gel (Sigma Aldrich CHEMIE GmbH, Germany) in 1x TBE (89 mM Tris base, 89 mM Acid borique; 2 mM EDTA [pH 8.0]) buffer at room temperature using gradients of 10V/cm.For gel analysis, 10 µl of the product was loaded in each gel plot.1Kb DNA Ladder from NEB was used to determine the fragment sizes.Finally, results were visualized after staining the gel in 0.5mg/ml ethidium bromide.
Following PCR analysis, a multiplex PCR assay was conducted for molecular characterization of the capsular antigens of P. multocida using primer sets specific for serogroups A, B, D, E, F [15,16].The serogroup-specific primer sets were identified according to the following criteria: (i) primer sets located within genes established as unique for each of the five serogroups (hyaD, bcbD, dcbF, ecbJ, and fcbD) (table 1), and (ii) amplicon length sufficient to allow clear size discrimination.The multiplex PCR mixture contained each primer within the six primer sets at a concentration of 0.4µM, 1x Standard Taq (Mg-free), 2 mM MgCl2, each deoxynucleoside triphosphate at a concentration of 0.2mM, 2U of Taq DNA polymerase.The PCR cycling conditions consisted of an initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30s, extension at 72°C for 60 s, and a nal elongation at 72°C for 5 min.The amplified products were separated by electrophoresis in 1% agarose gels and visualized by ethidium bromide staining.
All ampli cation reactions were performed using Gene Amp 9700 PCR system (Applied Biosystem).
Sequencing and phylogenetic analysis DNA was extracted from fresh bacterial cultures, using ISOLATE II Genomic DNA Kit (Bioline), and resuspended in 100µl of nuclease free distilled water.Five microliters of the extracted DNA were ampli ed using OneTaq DNA Polymerase (neb) Kit.The reaction was carried out in 50 µl using 16S ribosomal RNA [17] and rpoB [18] gene primer sets (table 2) under the following conditions: denaturation for 5 min at 95°C and 35 cycles of ampli cation as follows: denaturation at 94°C for 30s, annealing at 54°C for 30 s, extension at 72°C for 40 s, and nal extension at 72°C for 5 min.The length of the products was 1400 bp for 16S ribosomal RNA gene and 539 bp for rpoB gene.The resulting PCR products were puri ed using gel extraction kit (QIAEXII) according to the manufacturer instructions.The puri ed products were then sequenced by GATC Biotech Company.
The sequences were aligned and compared with publicly available sequences representing genetic diversity among Pasteurella multocida strains of avian, bovine, ovine and porcine origin.A phylogenetic tree was constructed using the neighbour-joining method and Kimura two-parameter model in MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets [19].

Pathogenicity of the isolates in laboratory animals (mice and guinea pig)
In experiment 1, pathogenicity of the ve P. multocida isolates was determined in ten 5 weeks old BALBc mice per strain (n=20) and six guinea pigs (n=12) by intra peritoneal inoculation of 0.5 ml of bacterial suspensions (1x10 9 cells/ml) grown in nutrient broth.The inoculated mice and guinea pig were observed for up to 3 days post inoculation for mortality.

Pathogenicity in sheep
Pathogenicity of the strain (S14) was determined in four 5 months-old healthy sheep.Before infection, nasal, conjunctival and rectal swabs as well as whole blood samples were collected to check the Pasteurella multocida status in sheep.Two sheep (G1) were inoculated with 4 mL subcutaneously, 2 ml orally and 2 ml by intranasal route of bacterial suspension (1.0 10 9 CFU/ml) and two other sheep (G2) inoculated with lower concentration (1.0 10 7 CFU/ml).Rectal temperatures were monitored daily during the 7-day post infection (p.i).Clinical signs such as respiratory rate, respiratory symptoms, feed intake and general behaviour were evaluated throughout the study.Oral and nasal swabs were collected from infected sheep at days 3, 6 and 9 pi.At day 7 pi, animals were autopsied and their lungs, mesenteric lymph nodes, liver and spleen were collected for bacteriological and PCR analysis.

Clinical cases
Lung tissues were obtained from dead animals: 3 sheep, 1 calve and 1 goat, from distinct herd and regions of Morocco.At necropsy, animals had lesions compatible with pneumonic disease including: pleurisy with abundant and brinous pleural effusion, pericarditis with gelatinous exudate on the pericardium, bronchopneumonia with increased volume of the lungs which are edematous and haemorrhagic and exudative mucopurulent in ammation in the respiratory tract.

Culture and biochemical identi cation
From the 5 eld specimens isolated from sheep, calve and goat(S11, S12, S13, S14, S15), P. multocida was recovered from only two tissue lung samples of sheep (2/5).All isolates with positive reactions for catalase, oxidase, and indole production, were con rmed as P. multocida by API 20NE.

Molecular detection and genotyping of Pasteurella
The 5 eld specimens isolated were used for detection of P. multocida, threalosi and M. haemolytica by PCR assay.From these specimens PCR showed 2 positive results (S13 and S14) and only P. multocida was detected.Both Pasteurella multocida strains were typed as serogroup A by the multiplex capsular PCR assay.

Sequencing and phylogenetic analysis
Phylogenetic analysis using ARN 16S gene among the two P. multocida strains (S13 and S14) demonstrated that S13 isolate was closely related to both an England strain (PM30) (99.5%) [20] isolated from bovine host and to a porcine isolate (IVRI) originated from India (99.7 %) [21].The S14 isolate was related to the Chinese PmCQ6 strain isolated from bovine host and evaluated as low virulent strain [22].
The RpoB gene analysis showed a similarity (99.38%) between the strain S13 and 964 isolated from goose host and originated from Hungary [23].The strain S14 was related to the Chinese strain PM-L1706 (99.33%) isolated from a chicken host (MG813902).
Determination of virulence P. multocida isolates in laboratory animals (mice and guinea pig) In experiment 1, the pathogenicity of 2 isolates was estimated by infecting 2 groups of 10 mice and 6 guinea pigs with 0.5 mL of a log phase culture containing infective doses of 1.10 9 CFU.Inoculated animals were observed for 3 days post inoculation for mortality.All mice infected with the strain S14 died in a short period of 15h, while those infected with the strain S13 showed 60% of mortality within 24h.
Similarly, in guinea pig the strain S14 registered a higher percentage of mortality with a short time period (66%, 24h) compared to the strain S13 (33%, 44h).However, it seems that the virulence of these two strains is more pronounced in mice than in guinea pig (table 4).
In experiment 2, virulence of the strain S14 was determined in a group of 22 mice based on mortality and time of death associated with different culture dilutions (table 4).All mice died at tested dilutions with a time period that ranged from 6h to 67h.

Clinical signs
Sheep were allowed to acclimate to the laboratory environment for a period of 2 weeks prior to experimental infection with Pasteurella multocida.During that time, all experimental animals were healthy and free of disease, with normal rectal temperatures.Animals of G1 (17 and 18) injected with the bacterial suspension, suffered from dyspnea, coughing, nasal discharge and developed a long hyperthermia (above 39°C) for 6 days: a peak was noticed between day 1 and day 3 p.i (40.5°C) for animal 17 and at day 2 pi (40.2°C) for animal 18 ( gure 3).Animals of G2 did not show any clinical symptoms but presented a hyperthermia (above 39°C) for 4 days: a peak was registered at day 1 pi (40°C) for animal 986 and between day 1 and 2 pi (40.6°C) for animal 987 ( gure 3).

Respiratory symptoms
Both groups of animals presented an important increase in respiratory frequency between day 1 and day 6 pi.A peak was registered at day 1 for animal 17 (50 breath/min) and 986 (55 breath/min), day 3 for animal 18 (60 breath/min) and day 6 for animal 987 (40 breath/min) ( gure 4).

Post mortem lesions
At post mortem at day 7 pi, sheep of G1 inoculated with 1.0 10 9 CFU presented lesions in both left and right lung, hypertrophy of mesenteric lymph nodes and intestinal congestion ( gure 5).For G2 animals, no lesions were observed at post mortem.

Bacteria excretion and tissue charge
In G1 inoculated with 1.10 9 CFU, Pasteurella has been detected by PCR in nasal swabs and lung in both animals 17 and 18 between d3 and d7 pi.In orotracheal swabs, the genome has been detected only in animal 18, between d6 and d7 p.i.In G2 inoculated with 10 7 CFU, no Pasteurella has been detected in nasal and orotracheal swabs.In the other organs (spleen, liver, mesenteric and pulmonary lymph nodes), no pasteurella has been detected for both groups G1 and G2.

Discussion
To date, no scienti c studies were published on the isolation and identi cation of Pasteurella in Morocco.This bacterium is usually associated with viral diseases such as PPR which is endemic in North Africa.In this region, the prevalence of different serotypes still unknown, however, eld veterinarian reports on clinical cases and lesions observed in abattoirs, suggest circulation of Pasteurella or Manheimia among ruminant populations.
No vaccination compaigns against pastereurella are conducted in small ruminant population.Recently, two vaccines against pasteurellosis combined to clostridia have been introduced in the market with satisfactory results in the eld, and a clear difference was observed between vaccinated and unvaccinated ocks regarding zootechnique performance and health general status.
The present study aimed to determine circulating Pasteurella strains in small ruminants and assess their pathogenicity with a main objective to develop a speci c vaccine that could match to the eld needs.
In our study we collected ve samples from dead animals in the eld with clinical symptoms similar to Pasteurella.Only two of the 5 strains were identi ed as Pasteurella multocida A suggesting that this serotype is dominant in the eld, but other agents maybe incriminated since 3 isolates need to be identi ed.Indeed, studies previously conducted in other African countries, showed the predominance of Pasteurella multocida in sheep from Senegal [24], however, in Sudan and north Cameroun, M. Heamolytica was the most isolated serotype in small ruminants population with an important antigenic variety [25][26][27].
The two Pasteurella isolates ful lled biochemical and molecular criteria of identi cation.These 2 strains, were genetically analysed using partial 16S rRNA and rpoB gene sequence comparison, the most used genes for Pasteurella species identi cation and phylogeny [28].Phylogenetic study revealed that both P. multocida A ovine isolates (S14 and S13) are related to strains from different host species (bovine, porcine and avian), suggesting that this type is not speci c to one animal species and may cause haemorrhagic septicaemia in pigs, cattle and avian hosts; which is in accordance with other previous phylogenetic studies [29][30][31].Thus, the transmission of bacteria between different host species, may constitute a factor in the population biology of P. multocida [32].
Pathogenicity on laboratory animals showed high sensitivity of mice to Pasteurella infection and the higher virulence of the strain S14 compared to S13 in both mice and guinea pig, that showed different mortality time depending on the strain and the inoculated dose.This suggest that the S14 strain may have caused severe clinical symptoms and important mortalities in the eld.Furthermore, kinetic infection study with S14 strain in mice showed that mortality occurs rapidly after 6h using the dose 109 and there was marginally later onset with lower doses (9h to 67h), which is in accordance with different studies having assessed P. multocida pathogenicity in mice [33][34][35].Also, results of this experiment suggest that mice may constitute a suitable challenge model for Pasteurella vaccine evaluation.This results were con rmed by other studies attempting to nd an alternative animal model for P. multocida and haemorrhagic septicaemia in cattle and avian pasteurellosis [33,[35][36][37], but more investigations are needed to develop this model.
Regarding guinea pigs, pathogenicity was reduced compared to mice, but all animals reacted positively to the infection by both S14 et S13 isolates with an advantage of S14.This species could also be used to test vaccine potency by challenge.
Clinically, sheep infected with S14 appeared to have suffered the most severe illness and presented lesions similar with the natural disease varying only in intensity.The infectious dose, 10 9 seems to be adequate to reproduce the disease in sheep.However, observed symptoms were much more severe in one sheep than in another that showed only lung lesions and mild symptoms.This could be attributed to an interaction between a number of factors such as burden of infections, individual defence mechanism, and immunity level.

Conclusion
Our study suggests that multocida A is frequent in the country with high incidence and economical losses.However, more investigations and survey studies are required to conclude on the predominance of Pasteurella serotypes in small ruminants population.To control the disease, vaccination remains the most e cient tool, particularly with vaccines based on local strains.

Ethics approval and consent to participate
Animal experiments were carried out in accordance with the international guidelines for care and handling of experimental animals, as the protocol has been submitted and approved by the Internal Ethic Committee "The internal ethic committee for animal experiment, MCI santé animale".Tables Table 1. of oligonucleotides used in the P. multocida multiplex capsular PCR typing assay.
Breathing rate of infected sheep G1 and G2 with Pasteurella multocida strain (S14)

2 :
Sequences of oligonucleotides used for P. multocida isolates sequencing Gene Sequences RpoB GCA GTG AAA GAR TTC TTT GGT TC GTT GCA TGT TNG NAC CCA T 16S ribosomal RNA AGA GTT TGA TYM TGG C GYT ACC TTG TTA CGA CTT

Figures Figure 1
Figures

Table 4 :
Percentage of mortality and time of death in mice (n=22) infected with different dilutions of P. multocida S14 culture.