Potential Treatment Options in a Post-antibiotic Era.

Following the Golden Age of antibiotic discovery in the previous century, the rate of antibiotic discovery has plummeted during the past 50 years while the incidence of antimicrobial resistance is ever-increasing. Presently, humankind is forced to address a major public health threat in the form of multiple drug resistance and urgent action is required to halt the advent of a post-antibiotic era. This chapter aims to draw the attention to the escalating global crisis of antimicrobial resistance fueled by the irresponsible use of antibiotics in healthcare and animal production sectors. The merits of alternative prevention and treatment options, including vaccines, herbal products, bacteriophages, and improved biosecurity measures are also discussed.

Lactococcus garvieae: an emerging bacterial pathogen of fish.

Lactococcus garvieae is the causative agent of lactococcosis, a hyperacute, haemorrhagic septicaemia of fish. This bacterium is also considered an emerging zoonotic pathogen, as reports of human infection are increasing. Significant economic loss in aquaculture is suffered as a result of lactococcosis, as numerous freshwater and marine species of commercial interest are affected. Development of antibiotic resistance in L. garvieae to several chemotherapeutic agents complicates and restricts treatment options. Effective, sustainable treatment and prevention options are thus needed, but progress is impeded by the lack of knowledge concerning several aspects of the disease and the pathogen. This review aims to present the latest research on L. garvieae, with specific focus on pathogenesis, virulence factors, risks associated with chemotherapeutic administration and possible control options.

Control of eight predominant Eimeria spp. involved in economic coccidiosis of broiler chicken by a chemically characterized essential oil.

AIM: To control eight most predominant Eimeria spp. involved in the economic disease of coccidiosis in broiler chicken, by a chemically characterized essential oil of eucalyptus and peppermint. METHODS AND RESULTS: The experimental design consisted of 160 day-old-broiler chicks, divided into four equal groups (G1 , G2 , G3 and G4 ), with 40 birds per group. Each group was divided into four equal subgroups. Birds in G1 were deprived of essential oil treatment and of Eimeria challenge. Birds in G2 were unchallenged, and administered the essential oil in drinking water at 0.69 ml kg(-1) body weight. Birds in G3 were untreated with essential oil, and each of its four subgroups was challenged at a different age (14, 21, 28 and 35 days). Birds in G4 were treated with essential oil, and challenged in the same manner as for G3 . Equal number of birds from all subgroups (n = 10) were sacrificed at the sixth day after the time allocated for each challenge. The 6 day incubation period post challenge resulted in respective mean per cent weight increase in G2 and G1 birds equivalent to 57.8 and 53.1% (P < 0.05). In addition, the essential oil improved the per cent weight increase in challenged birds (54.6%) compared to the challenged-untreated birds (18.6%) (P < 0.05). The mean feed conversion, mortality, intestinal lesion scores and oocyst counts were significantly reduced in the challenged-treated birds compared to the challenged-untreated birds (P < 0.05). CONCLUSIONS: The results support the hypothesis of using the essential oils of eucalyptus and peppermint to control the most prevalent Eimeria spp. involved in coccidiosis of broiler chicken, helping in improvement of their production, alleviation of lesions and reduction in intestinal oocyst counts. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information about the possibility of using this blend of essential oil as a coccidiostat for the protection of broiler chickens against the prevalent eight Eimeria spp. of coccidiosis.

Modulation by essential oil of vaccine response and production improvement in chicken challenged with velogenic Newcastle disease virus.

AIM: To evaluate the impact of Eucalyptus and peppermint essential oils on immune modulation and production of broiler chicken challenged with a molecularly characterized velogenic NewCastle disease virus (vNDV). METHODS AND RESULTS: The experimental design included five treatments with three replicate pens/treatment comprised of 12-day-old broilers chicks/replicate. The five treatments included a positive challenge control (non-NDV vaccinated/nonessential oil treated/challenged) (NNEOC), a negative challenge control (NDV vaccinated/essential oil treated/unchallenged) (VEOU), a non-NDV vaccinated/essential oil treated/challenged (NEOC), a NDV vaccinated/nonessential oil treated/challenged (VNEOC) and a NDV vaccinated/essential oil treated/challenged (VEOC). The lowest mean survival rate (0·0%) and lowest production performance were obtained by the positive challenge control, while the best mean survival (93·3%) and average body weight (2649 g) were obtained by the negative challenge controls (P < 0·05). Among the three others challenged treatments, the best mean survival (79·2%), highest mean body weight at 42 days of age (2445 g), the lowest feed conversion ratio (1·60) and the highest serum conversion immunopotentiation at 35 days of age determined by ELISA and hemagglutination titres were obtained by the VEOC birds compared with respective means obtained by birds of the NEOC and VNEOC treatments (P < 0·05). CONCLUSIONS: The results supported the possibility of using the essential oils of Eucalyptus and Peppermint in broilers to immunopotentiate the response to vaccination against velogenic NDV, helping in significant improvement of survival and production. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information about the potential use of essential oils of eucalyptus and peppermint that can be exploited as commercial immunopotentiators for the protection of NDV-vaccinated broiler chickens against economic velogenic NDV.

Multiplex polymerase chain reaction for screening avian pathogenic Escherichia coli for virulence genes.

Colibacillosis is a disease in poultry caused by avian pathogenic Escherichia coli (APEC) strains which leads to great economic losses in the poultry industry. These E. coli strains contain various virulence genes which grant the bacteria the ability to proliferate in the poultry host and cause disease. Many genes which can contribute to virulence have been identified and can be used to screen E. coli strains to infer pathogenicity and aid in the identification and classification of APEC. Multiplex polymerase chain reaction methods were designed and optimized to rapidly detect 18 different virulence genes in E. coli strains that were isolated in South Africa and Zimbabwe from various sources, including from chickens showing signs of colibacillosis. Virulence gene profiles were constructed for each E. coli isolate from the multiplex data for the comparison of the colibacillosis isolates with the other isolates. The South African E. coli isolated from chickens with signs of colibacillosis showed higher virulence gene prevalence in comparison with the Zimbabwean and other samples except those isolated from chicken faeces. The multiplex polymerase chain reaction designed in the present study successfully screened E. coli isolates for various APEC-related virulence genes, including genes recently described in the literature.

Study on the efficacy and safety of different antigens and oil formulations of infectious coryza vaccines containing an NAD-independent strain of Avibacterium paragallinarum.

The present study was designed to assess and compare three different formulations of the new Onderstepoort infectious coryza (IC) quadrivalent vaccine, which contain an NAD-independent strain of Avibacterium paragallinarum (previously known as Haemophilus paragallinarum), and a commercial IC vaccine, not containing an NAD-independent strain, for their safety and ability to protect chickens of varying ages against virulent challenges with four different serovars of A. paragallinarum, including the NAD-independent strain of the C-3 serovar. Four groups of 140 chickens each were vaccinated at the age of 17 weeks and revaccinated at the age of 19 weeks with each of the four vaccine formulations. A similar sized group of non-vaccinated chickens was used as control. Two rounds of challenge were conducted: a group of chicken in each vaccination group was challenged between 31 and 35 weeks of age, while another group was challenged between 51 and 55 weeks of age. The "in-contact" challenge model was used in this experiment. For each vaccination group, the four challenge strains representing four local serovars were used in each challenge round. The efficacy of the vaccines was compared based on overall protection levels obtained and the duration of protection. The safety of the different vaccines was determined by the severity of post-vaccination reactions. The need for the incorporation of the NAD-independent strain in the vaccine was evidenced by the low protection level against NAD-independent challenge recorded in the group of birds vaccinated with the commercial vaccine. The results obtained confirmed not only the variation in virulence of different South African serovars, with serovar C-3 being the most virulent and serovar B having almost no virulence but also the age related increase in susceptibility. The importance of a suitable formulation of the vaccine is discussed.

Haemophilus paragallinarum haemagglutinin: role in adhesion, serotyping and pathogenicity.

It is suggested that Haemophilus paragallinarum requires at least three haemagglutinins for adhesion during infection. This paper reports the partial purification and characterization of the HA-L haemagglutinin from H. paragallinarum strain 46-C3, a heat sensitive, trypsin sensitive haemagglutinin that has been shown to be the serovar specific haemagglutinin in this organism. Using the pl and molecular mass obtained, it was shown that this protein shares similarities with other types of adhesins found in Gram-negative bacteria. The haemagglutination assay conditions were optimized at pH 7.5 at 37 degrees C. It was also shown that activity is enhanced by the addition of Ca2+ and Mn2+ ions.

Genetic diversity of the Rep gene of beak and feather disease virus in South Africa.

A study on the genetic variation of Beak and feather disease virus (BFDV) isolates in South Africa was performed by amplifying and sequencing a region within the ORF1 of the genome. Six different BFDV isolates were found in 15 psittacine species from 6 regions within South Africa, representing three unique isolates and three isolates that clustered into a budgerigar lineage (BG) previously described.

Beak and feather disease virus haemagglutinating activity using erythrocytes from African Grey parrots and Brown-headed parrots.

Psittacine beak and feather disease (PBFD) is a common viral disease of wild and captive psittacine birds characterized by symmetric feather loss and beak deformities. The causative agent, beak and feather disease virus (BFDV), is a small, circular single-stranded DNA virus that belongs to the genus Circovirus. BFDV can be detected by PCR or the use of haemagglutination (HA) and haemagglutination inhibition (HI) assays that detect antigen and antibodies respectively. Erythrocytes from a limited number of psittacine species of Australian origin can be used in these tests. In South Africa, the high cost of these birds makes them difficult to obtain for experimental purposes. Investigation into the use of erythrocytes from African Grey parrots and Brown-headed parrots yielded positive results showing the haemagglutinating activity of their erythrocytes with purified BFDV obtained from confirmed clinical cases of the disease. The HA activity was further confirmed by the demonstration of HI using BFDV antiserum from three different African Grey parrots previously exposed to the virus and not showing clinical signs of the disease.

Effects of differences in virulence of different serovars of Haemophilus paragallinarum on perceived vaccine efficacy.

The virulence of four South African field isolates of NAD-dependent Haemophilus paragallinarum and two field isolates of NAD-independent H. paragallinarum has previously been tested in unvaccinated chickens. In this study, the disease profiles caused by the NAD-dependent isolates of H. paragallinarum in vaccinated chickens were studied. It was shown that the clinical signs induced in the vaccinated chickens were substantially less severe than were those in unvaccinated chickens, as was expected. However, due to the high virulence of the serovar C-3 isolates, clinical signs in the vaccinated chickens challenged with this isolate were still detected. These were as severe as those occurring in unvaccinated chickens challenged with serovar B-1 isolates. Although the clinical signs induced in unvaccinated birds challenged with serovar A-1 were more severe than those occurring when vaccinated birds were challenged with serovar C-3, the overall disease profiles were similar. Substantial clinical signs were recorded in vaccinated birds challenged with serovar C-3. This could be interpreted as vaccination failure if the disease profile obtained in unvaccinated birds is not considered. It was found that a high level of protection was provided by this vaccine against challenge by serovar C-3. The high virulence of this serovar resulted in the development of clinical signs in vaccinated birds. These findings could possibly explain the large number of so-called vaccination failures that are reported in South Africa.

The testing and modification of a commercially available transport medium for the transportation of pure cultures of Haemophilus paragallinarum for serotyping.

Haemophilus paragallinarum, the causative agent of infectious coryza in poultry, is an extremely fastidious organism requiring specific growth conditions for isolation. For complete control of the disease in regions where more that one of the serovars of the different serogroups occurs, it is essential that the bacterium causing the problem be isolated and serotyped. This work describes the modification and testing of transport media, which will ensure the survival of the causative agents in suspected infectious coryza cases for transport to a laboratory where the bacterium can be isolated and serotyped. The various transport media used are based on commercially available Amies Transport Medium supplemented with the different supplements used for the growth of H. paragallinarum. It was established that the bacterium remains viable for up to 18 days in Amies Transport Medium containing all the supplements when stored at 4 degrees C or 37 degrees C. At room temperature or 25 degrees C, there was no difference in the survival of H. paragallinarum in commercial Amies Transport Medium (without charcoal) and Amies Transport Medium with supplements.

Genetic organisation of the capsule transport gene region from Haemophilus paragallinarum.

The region involved in export of the capsule polysaccharides to the cell surface of Haemophilus paragallinarum was cloned and the genetic organisation determined. Degenerate primers designed from sequence alignment of the capsule transport genes of Haemophilus influenzae, Pasteurella multocida and Actinobacillus pleuropneumoniae were used to amplify a 2.6 kb fragment containing a segment of the H. paragallinarum capsule transport gene locus. This fragment was used as a digoxigenin labelled probe to isolate the complete H. paragallinarum capsule transport gene locus from genomic DNA. The sequence of the cloned DNA was determined and analysis revealed the presence of four genes, each showing high homology with known capsule transport genes. The four genes were designated hctA, B, C and D (for H. paragallinarum capsule transport genes) and the predicted products of these genes likely encode an ATP-dependent export system responsible for transport of the capsule polysaccharides to the cell surface, possibly a member of a super family designated ABC (ATP-binding cassette) transporters.

Limitation of the spread and impact of infectious coryza through the use of a continuous disinfection programme.

The effect of a continuous disinfection programme, using the non-toxic disinfectant Virukill, in layers, on the spread and impact of infectious coryza, caused by Haemophilus paragallinarum was evaluated. In this experiment, both unvaccinated layers and layers vaccinated against infectious coryza were used. Duplicate smaller groups of vaccinated and unvaccinated chickens were challenged with different serovars of both NAD-dependent as well as NAD-independent isolates of Haemophilus paragallinarum. One group of chickens challenged with each of the different becterial serovars was treated with the continuous disinfection programme, while the other group remained as the untreated controls. The clinical signs of infectious coryza were evaluated over a period of 20 days in each group. The egg production over this period was also evaluated. It was found in all experimental challenges, that the severity of the symptoms was reduced in the birds receiving the continuous disinfection programme. The drop in egg production was also found to be less severe in the treated groups when compared to the untreated control groups. The duration of infection was found to be either unchanged, or shorter in the birds treated with the continuous disinfection programme. In none of the experimental challenges was the duration or expression of clinical signs of IC increased due to the continuous disinfection programme.

Psittacine beak and feather disease virus in budgerigars and ring-neck parakeets in South Africa.

Psittacine beak and feather disease (PBFD) is a common disease of the psittacine species and is caused by the psittacine beak and feather disease virus (PBFDV). In this study the occurrence of the disease in ring-neck parakeets and budgerigars in South Africa suffering from feathering problems, using polymerase chain reaction as a diagnostic test was investigated. The genetic variation between viral isolates was also studied. Results indicate that PBFDV can be attributed to being the cause of feathering problems in some of the ring-neck parakeets and budgerigars in South Africa. Genetic variation of isolates occurs between species and individuals. A cheap and easy to use method of blood sample collection on filter paper for diagnostic purposes was also evaluated. It proved to be less stressful to the birds and did not inhibit further processes.

Evidence of possible evasion of protective immunity by NAD-independent isolates of Haemophilus paragallinarum in poultry.

An indication of the ability of NAD-independent variants of Haemophilus paragallinarum to evade the immune system has been obtained from data obtained from several experiments. Firstly, it was noted that there was a difference in the serovar distribution between the NAD-dependent isolates in the 1990s and the NAD-independent isolates, as there was a significant decrease in the incidence of serogroup A NAD-dependent isolates. This can possibly be attributed to the extensive use of vaccines. On the other hand, most of the earlier NAD-independent isolates were serovar A. This is a possible indication of evasion of the protective immunity by the NAD-independent isolates. Further evidence of possible evasion of the protective immunity was obtained from results obtained when different isolates, both NAD dependent and NAD independent, were tested with a panel of monocional antibodies (Mabs). The V1 Mab reaction pattern was only seen in the reference strain 0083 among all of the NAD-dependent isolates tested in South Africa. This Mab was, however, found to react with some of the NAD-independent isolates. Furthermore, the isolation of NAD-dependent isolates in Australia which react with the V1 Mab also suggest possible evasion of the protective immunity by the NAD-independent isolates as no vaccines containing strain 0083 are used in Australia. In order to investigate the hypothesis of immune-evasion by NAD-independent H. paragallinarum, vaccinated and unvaccinated chickens were challenged with a NAD-independent serogroup C isolate. As a control, chickens were also challenged with NAD-dependent H. paragallinarum of the same serogroup. The results obtained indicate that there is no significnat difference in the disease profiles obtained in vaccinated and unvaccinated chickens challenged with the NAD-independent isolate, thus providing further evidence of evasion of the productivity immunity by the NAD-independent isolates. The ability of the NAD-independent isolates to evade the immune system suggests that a different vaccination strategy, or alternative control methods may be needed for the control of IC caused by these isolates.

Continuous disinfection as a means to control infectious diseases in poultry. Evaluation of a continuous disinfection programme for broilers.

A full continuous disinfection programme, consisting of disinfection during cleanout of poultry houses prior to placement of chickens, disinfection of the drinking water and spray disinfection of the birds during production was evaluated in broilers under experimental condition as well as under field conditions. Under controlled conditions, the experimental design consisted of three groups, two of which were control groups. Each group comprised 300 chickens. In one of the control groups, no disinfection of the pens was undertaken prior to the placement of the chickens. In the other control group, disinfection of the pens prior to placement of the birds was carried out using a glutaraldehyde-based product. In the test group, disinfection prior to placement was done. The drinking water of these birds was treated continuously and the birds were sprayed with a non-toxic disinfectant during production. Production parameters, such as growth rate, feed conversion ratio and feed consumption, of the birds in the three groups were monitored. In addition, all mortalities in the different groups were recorded and classified into diseases of an infectious nature, non-infectious nature and unknown category. Bacterial counts were also done on a weekly basis from the different pens. In this experiment, it was shown that the full continuous disinfection programme resulted in a lower number of mortalities caused by infectious agents as well as a reduction in the bacterial counts in the pens treated with the full continual disinfection programme. The full continuous disinfection programme was also tested on a commercial poultry farm in the Northern Cape Province of South Africa. Two production houses of 3,500 birds were randomly selected as test houses for the full continuous disinfection programme. Another similar house, which received day-old chicks from the same batch as the other two houses, was selected as the control house; it received the routine disinfection procedure prior to placement of the chicks. During the course of this experiment, a severe outbreak of Newcastle disease was experienced on this farm. It was demonstrated that, in the face of this severe challenge, the full continuous disinfection programme controlled the spread of the disease in both the houses where it had been applied at a stage when in every other house (including the control house) on the farm birds were suffering very high mortalities.

Virulence of South African isolates of Haemophilus paragallinarum. Part 3: experimentally produced NAD-independent isolate.

A NAD-dependent isolate 46 (C-3) of Haemophilus paragallinarum, which was previously demonstrated to be of high virulence, was transformed to NAD independence using a plasmid isolated from a naturally occurring NAD-independent isolate of H. paragallinarum. The transformation was performed by two different methods and the identity of all of the isolates, before and after transformation was confirmed using a H. paragallinarum-specific PCR test. The transformed NAD-independent serovar C-3 isolate and the wild-type serovar C-3 isolate were used to experimentally infect vaccinated layer chickens. It was shown that the transformation to NAD independence significantly altered the virulence of the serovar C-3 isolate that was used in the transformation experiment. The mechanisms responsible for a decrease in virulence are not clear, but may be related to the pathology of the transformed isolate in the sinus of the chickens.

Isolation of serovar C-3 Haemophilus paragallinarum from Zimbabwe: A further indication of the need for the production of vaccines against infectious coryza containing local isolates of H. paragallinarum.

Various isolates of Haemophilus paragallinarum, collected from a severe outbreak of infectious coryza in poultry from Zimbabwe, were serotyped and were found to belong to serovar C-3. Previously, isolates were serotyped using polyclonal antiserum produced against serogroup reference strains (0083 for serogroup A, 0222 for serogroup B and Modesto, or H-18 for serogroup C) of H. paragallinarum. In this case, polyclonal antiserum produced against these reference isolates were used, as well as polyclonal antiserum that has been raised specifically against the serovar C-3 isolate 46 C-3. When using the latter serum at a 1 in 50 dilution, no cross-reaction with other members of serogroup C were found. The severity of the disease outbreak in Zimbabwe, the vaccination history of the infected flocks on the sites and the isolation of the uniquely southern African serovar C-3, further highlights the need for vaccines composed of local isolates to control infectious coryza in regions where vaccination failures occur.