El control de la gestión y sus elementos

The research presented here on the causes of neonatal mortality in NZSLs has highlighted two interesting aspects. First, it showed that the main causes of mortality at Sandy Bay Beach on Enderby Island were being diagnosed in the same chronological order, for every breeding season. Secondly, it underlined the relative vulnerability of this NZSL population to unusual pathogens such as Klebsiella pneumoniae. Uncinariosis was considered to be a primary cause of death in about 13% of pups but all were found to be infected with hookworms. Consequently, determining the role of these parasites in neonatal mortality was complex but a comparative study involving artificially parasite-free and naturally infected pups allowed the investigation of the effect of hookworms on pup health.

I. Hookworms and pinniped neonates

Uncinaria spp. was the only endoparasite found in NZSL pups over the period of

study, which is consistent with what has been observed in other pinnipeds (Lucas, 1899; Baylis, 1947; Olsen, 1952; Dailey and Hill, 1970; Botto and Mañé-Garzón, 1975; Berón-Vera et al., 2004). The hookworm species described in NZSLs presented some morphometric differences from the two other species already reported in pinnipeds (Baylis, 1933, 1947) and it is suggested to be at least a separate strain if not a different species. This raises the possibility that other epidemiological aspects might also be different. However, molecular characterisation is required to fully determine if the hookworms infecting NZSL pups are a novel species.

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Studies undertaken in the current research on the life cycle of hookworms in NZSLs have shown similarities with Uncinaria lucasi in northern fur seals and California sea lions (Lyons, 1963; Lyons et al., 2000a). Although based on observations in three NZSL newborns, it is evident that these pups were ingesting L3s through the colostrum.

Further evidence supporting transmammary transmission was the finding of ten larvae of similar size and morphology in the mammary glands of lactating adult females.

Faecal examinations demonstrated that pups were shedding large numbers of hookworm eggs on the beach from three weeks of age indicating the minimum prepatent period would be about this long. It is hypothesised that these eggs will then develop into free-living infective larvae that will infect all NZSLs. These, in turn, would be the source of transmmary transmission for future generations of pups. Evidence to support this hypothesis was the successful culturing of ensheathed L3s in the field and

the finding of similar larvae (without a sheath) in the subcutaneous tissues of NZSLs (especially in pups, in an adult male and in non lactating females). The general size of these L3 was consistent with each other and with reports of similar stages of Uncinaria

lucasi in northern fur seals (Lyons, 1963; Lyons and Keyes, 1978). The finding of

larvae in the subcutaneous tissue of NZSL female pups suggests these may be a source of transmammary infection for their offspring.

It is not known when pups expel hookworms from their intestines or what mechanisms are associated with this phenomenon. The immune response developed by the host towards parasites is part of a complex dynamic between the two parties, with the ideal outcome being a balanced relationship, and the two extremes resulting in disease or in the expulsion of parasites. Necropsy of NZSL yearlings has shown that they no longer harbour intestinal infection with hookworms.

One way to maintain the hookworm life cycle on the rookery is the possibility that larvae could overwinter in the sand. In the current study, such larvae were found in soil samples at the end of the breeding season but were not present in samples taken at the same sites at the beginning of the following season. However, this was not surprising as the sand at Sandy Bay Beach is washed away during the winter. This suggests that the only likely source of infection for future generations of pups on this rookery is the

hypobiotic larvae that reactivate during lactation. It is unknown whether all of these inhibited larvae reactivate at the next lactation or only some, implying that a single pool of dormant larvae would ensure that several generations of pups could become infected, without requiring reinfection. Studies in captive pinnipeds have shown that larvae could at least survive several years before being transmitted to the pup (Lyons and Bigg, 1983; Lyons and Keyes, 1984). Overall, the biology of Uncinaria spp. in NZSLs resembles that reported for Uncinaria lucasi in other pinniped hosts (Lyons et al., 2000a, 2000b).

Clinical pathology associated with hookworm infection in NZSL pups was similar to lesions reported in dogs infected with Ancylostoma caninum (Miller et al., 1971; Kalkofen, 1987). It mainly consisted of haemorrhagic enteritis with large amounts of frank blood in the intestinal lumen. However, contrary to the situation reported in dogs, there was no apparent relationship between burden and lesions in the NZSL. In addition, the present research did not succeed in showing any relationship between haematological parameters and the occurrence of hookworm infection. There was no evidence of anaemia in live infected pups. Why some pups succumbed and not others is at present uncertain.

Another possible pathological role of Uncinaria spp. in NZSLs was its effect on neonatal growth. Even though studies investigating the effect of parasitism on neonatal growth in wild animal species are very scarce, hookworm infection may be associated with weight loss, lack of appetite and growth delay in human neonates, children and in young domestic carnivores (Miller, 1971; Anderson, 2000; Kucik et al., 2004). In NZSL pups, hookworm infection would not impact on pup growth and survival (Chilvers et al., in preparation).

Some associations between pathogens happen to be synergistic: each species increases the pathogenicity of the other (Petney and Andrews, 1998). This may have been the case in NZSL pups infected with hookworms during the Klebsiella

pneumoniae epidemics. Parasites could have debilitated pups, and these may have

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II. Klebsiella pneumoniae epidemics

Klebsiella pneumoniae had not been identified as a cause of neonatal death prior to

2001/2002. During the 2001/2002 and 2002/2003 breeding seasons, this bacterium was cultured from a wide range of lesions and organs collected in NZSL pups at necropsy. In this respect, the high mortality events caused by K. pneumoniae during these two seasons fulfil the requirements to be described as an epidemic, in that there was a sudden onset and a high incidence of infection. Further evidence in support was the characterisation of K. pneumoniae as a single bacterial clone. Interestingly, it was different from K. pneumoniae cultured from adult NZSLs before and during the epidemics, although it was isolated from one adult male on the Otago Peninsula after the epidemics. The actual source of this particular clonal strain remains uncertain. From the limited testing undertaken, it was apparent that this K. pneumoniae clone was not resistant to common antimicrobial agents. This would suggest that it did not originate from humans as antibiotic resistance in human isolates of Klebsiella species is a common feature.

To investigate whether K. pneumoniae causing epidemics in NZSLs was a novel pathogen to the rookery, a serological survey was undertaken using WB. Results showed a high prevalence of anti-Klebsiella antibodies in adults, indicating that

Klebsiella species were circulating in the NZSL population. In contrast, the

seroprevalence in pups older than a month was only 16% regardless of whether it was an epidemic year or not, which suggested limited maternal transfer of passive immunity against Klebsiella pathogens. Even after the 2001/2002 and 2002/2003 epidemic seasons, there was no evidence of maternal transfer of anti-Klebsiella antibodies as these pups had a seroprevalence of 0%. This implies that young pups were not passively protected against K. pneumoniae infections. However, it was not possible to accurately determine the sensitivity of the WB test as NZSLs are endangered animals and experimental infections required to optimise test sensitivity were not possible. A limitation to these findings is also the lack of specificity of the WB. It was shown that this assay could not distinguish between two species of Klebsiella (Klebsiella oxytoca

and K. pneumoniae). Therefore this technique did not allow drawing a definite conclusion regarding the presence or the absence of this K. pneumoniae strain in the Sandy Bay Beach population prior to the epidemics.

Lesions associated with K. pneumoniae in NZSLs were dominated by arthitis, often in several joints, and septicaemia. A diverse range of lesions have been separately reported to be caused by K. pneumoniae in other animals, including humans (Giles et al., 1974; Jackson et al., 1980; Enurah et al., 1988; Coletti et al., 2001; Park et al., 2004). A feature of the disease in NZSLs was the broad spectrum of lesions that were caused by this pathogen and that were often observed in multiple organs of the same animal. Most

Klebsiella spp. infections reported in other animals are generally not associated with a

multi-systemic presentation of lesions.

III. Variability in the virulence of the opportunistic Klebsiella pneumoniae

Infections with opportunistic pathogens naturally occur in wild animals. Their functional immune system develops an adapted response and keeps the antigens in memory. Despite being specific, cellular immunity can protect individuals in the presence of slightly different pathogens. However, it is possible that a small genotypic difference between two strains of the same bacterial species generates a significant modification of its pathogenicity. It has been shown experimentally that the virulence of

K. pneumoniae causing metritis in horses was directly proportional to the “degree of

encapsulation” of the bacteria (Kikuchi et al., 1987). Even though antibodies against K.

pneumoniae may cross-react with other Klebsiellae, it is likely that the presence of a

particular type of capsular antigens requires highly specific antibodies. This hypothesis could explain the absence of immune protection of NZSL pups during the K.

pneumoniae epidemics, despite the presence of general anti-Klebsiella antibodies at

least in one in five pups. Highly virulent agents may owe their pathogenicity to the absence of highly specific immunity in the hosts.