Friday, May 28, 2010

Clinical findings and risk factors associated with the first report of Eimeria macusaniensis

In July 2005, the coccidian parasite Eimeria
macusaniensis was detected in New
Zealand alpacas. This paper describes the investigation
that followed the discovery.
Clinical findings and risk
factors associated with
the first report of Eimeria
macusaniensis in New Zealand
alpacas
Three coccidian species – Eimeria lamae, Eimeria alpacae and
Eimeria punonensis – have previously been reported in camelids in
New Zealand(1)(2)(3).
On 29 July 2005, a pathologist at Gribbles Veterinary Pathology
notified MAF of a suspected identification of E macusaniensis
in the faeces of a ten-year-old female alpaca.
The diagnosis was subsequently confirmed by a Biosecurity New
Zealand reference parasitologist.
At the start of the investigation, Biosecurity New Zealand
considered the coccidian to be a new incursion and, because of its
reputed pathogenicity(4)(5), declared it an unwanted organism under
the Biosecurity Act 1993. A measured response was initiated on 10
August 2005 with four objectives:
• to prevent spread of E macusaniensis by implementing interim
containment measures on the properties that received alpacas
from the affected import consignment,
• to complete a cross-sectional survey of camelids on affected
properties to detect those shedding E macusaniensis, and assist in
understanding the epidemiology of this parasite in New Zealand,
• to collect and assess case history data for ill alpacas in recent
import consignments,
• to commission Biosecurity New Zealand’s reference parasitologist
to review the literature on E macusaniensis, including its
potential pathogenicity (published in this edition(6)).
Interim containment
Movements of all camelids and their faecal material were restricted
on to and off the four properties suspected of having affected
animals. To enforce these measures each property was issued a
Restricted Place notice under s130 of the Biosecurity Act 1993.
AgriQuality Limited provided a Restricted Place Manager (RPM)
for each property to help manage the conditions of the Restricted
Place notice, and fulfil duties in accordance with MAF Standard
153 (Response Programs for Exotic Diseases of Animals, October
2004).
Cross-sectional survey
A cross-sectional survey was used to help elucidate the
epidemiology of E macusaniensis on affected properties. The survey
categorised the animals shedding E macusaniensis into four at-risk
groups:
• alpacas from the May import consignment,
• alpacas from previous import consignments,
• alpacas exposed to the May import consignment,
• alpacas not exposed to the May import consignment.
A fifth South Island farm with a single E macusaniensis positive
animal was identified through passive laboratory surveillance
around the time of the response, and was also included in the
cross-sectional survey. This property had been run as a closed flock
for a number of years and had no contact with recently imported
animals or the four infected places.
Faecal samples were collected from all alpacas older than three
months, with the exception of those close to parturition. The
census sampling was intended to provide a 95% confidence of
detecting at least a 2% prevalence of shedding animals in the
smallest of the five risk groups. A prevalence of 2% represents
the lower 95% confidence interval for prevalence determined in a
survey by Jarvinen(7) in the northwestern United Sates.
A sealable plastic bag was inverted and used as a glove for per
rectum collection of faeces. Samples were delivered the same day
to the laboratory and a faecal sedimentation technique used to
detect coccidial oocysts. Because of the large size of
E macusaniensis oocysts, faecal sedimentation is considered a more
sensitive technique than faecal flotation(7). All at-risk farms were
sampled in mid-August 2005.
Univariate analysis using chi-squared or Fisher’s exact methods
were used to explore the data for potential explanatory variables
associated with faecal shedding of E macusaniensis oocysts.
Analysis was undertaken using STATA 7.0 (Stata Corporation,
USA). A p-value less than 0.05 was considered significant.
Survey results
Prevalence of shedding
On the first four at-risk farms, 427 alpacas were sampled and 14
animals determined to be shedding (Table 1). A further 33 animals
were sampled on Farm 5 where a single animal had been identified
as shedding. Inter-farm prevalence of shedding ranged between
0.61% and 7.55% (Table 1, Fisher’s exact p = 0.033).
Table 1: The intra-farm prevalence of alpacas shedding Eimeria macusaniensis oocysts
on the five affected farms
Farm 1 Farm 2 Farm 3 Farm 4 Farm 5 Total
Date sampled
(2005)
12 August 15 August 12 August 15 August 25 and 29 August
Number sampled 164 1211 107 35 33 460
Positive
1
(0.61%)
81
(6.61%)
3
(2.80%)
2
(5.71%)
1
(3.03%)
15
(3.26%)
Fisher’s exact p-value = 0.033
1 Findings from a mob of 15 animals imported from Australia on to Farm 2 on 19 July were excluded from the summary figures above and in Table 1.
Table 2: Univariate analysis of risk factors for shedding of Eimeria macusaniensis
determined after investigation of five affected farms
Risk factor +ve
(prevalence of infection)
Risk factor -ve p-value
Risk factors for shedding
Univariate analysis was carried out to assess risk factors for
E macusaniensis shedding in individual alpacas. As noted in the
footnote to Table 1, findings from the mob of 15 animals imported
on to Farm 2 in July were excluded from this analysis. This was
because the prepatent period for E macusaniensis of 32-40 days
would mean infection in these animals would have taken place
before their entry into New Zealand(8). Six of the 15 (40%) animals
in this group were found to be shedding E macusaniensis oocysts.
Shedding was significantly associated with age (<12 months) and exposure to the May import consignment. Factors not significantly associated with shedding were sex, the import status of the animal, or whether the animal belonged to the May consignment (Table 2). There was no difference in the proportion exposed in the two age groups. Case history summaries Case history (clinical, necropsy and laboratory) data was available for four ill alpacas from recent import consignments. Eimeria macusaniensis was not implicated as the cause of death in a cria and two adults from the May import consignment, but was determined to be the cause of death of an alpaca in a July consignment. Hypothermia with terminal aspiration of stomach content was responsible for the death of the cria, and chronic liver disease was identified as the cause of death in the two adults from the May consignment. In one animal the aetiology of the liver pathology was not evident, and a toxic origin appeared likely in the remaining adult. The death of the alpaca from the July consignment was attributed to the severe gut pathology associated with infection by E macusaniensis. The key findings in this case are summarised below. This animal was an Australian bred adult female alpaca imported on to a Canterbury alpaca farm on 19 July 2005, after 30 days quarantine in Victoria, Australia. The farmer was experienced with alpacas, having successfully imported, bred and shown them for five years. On 14 August 2005, the farmer observed the animal to be in light condition (estimated condition score: 2-2.5) with loose (semi-solid) faeces. The farmer treated the animal with an oral coccidiocide (off-label use) but it died on 17 August. At MAF’s request, the farmer’s veterinarian carried out a postmortem, and collected samples for biochemical, faecal and histological analysis. Necropsy showed small intestinal gastroenteritis and enlarged mesenteric lymph nodes. The carcass was otherwise grossly normal. Significant histological lesions (Figures 1 and 2) were confined to the gastrointestinal tract. Occasional coccidial cysts were identified in the duodenum. The ileum was affected by a severe coccidial infestation with suppurative enteritis and multifocal ulceration. Large numbers of various stages (oocysts, schizonts, etc) of coccidia were present in the lamina propria. The number of crypts was reduced, with villi shortened and the sides of some villi covered in a flattened epithelium. The area of the basal lamina propria and muscularis mucosa had neutrophils and macrophage infiltrates with the reaction extending through the submucosa to affect the tunica muscularis in places. The rest of the submucosa had areas of oedema and widespread infiltration of neutrophils, macrophages and plasma cells. Multiple foci of ulceration were also present with ulcers filled with necrotic debris, bacteria and neutrophils. The spiral colon and large intestine had no significant lesions. Serum was collected at postmortem. Reduced blood albumen (20 g/l) and total protein (40 g/l) were consistent with a proteinlosing enteropathy. Elevated blood creatine (441 ╬╝mol/l), urea (33.6 mmol/l) and phosphate (5.71 mmol/l) were considered to have resulted from dehydration. Faecal sedimentation identified abundant (+++) E macusaniensis oocysts. Faecal culture for salmonella was negative. Discussion There have been conflicting reports in the literature of the importance of this protozoan. Clinical disease in individuals and groups appears to occur only sporadically, and when it does it is often associated with stress and/or poor management. Generally infection is subclinical with a low herd prevalence of faecal oocyst shedding (Jones J, UK, personal communication; Cebra C, USA, personal communication)(4). The range in herd shedding identified in the New Zealand investigation (0.61% to 6.61%) was comparable with that found in overseas studies(7)(9)(10). The range differed significantly among the New Zealand study farms (p = 0.033; Table 1). The cross-sectional survey identified age (less than 12 months) and exposure to the May import consignment as significant factors influencing shedding (Table 2). Eimeria macusaniensis is generally associated with clinical disease in young animals(11) and has been associated with death in neonatal alpacas in South America(5). A higher prevalence of shedding has been noted in young llamas(7)(8) although this has not previously been substantiated in alpacas. Management factors associated with importing animals are likely to increase susceptibility to E macusaniensis(12) and lead to increased rates of shedding with resultant contamination of the environment and exposure of other animals. The risk associated with recently imported animals is supported by the finding of a 40% prevalence of shedding in the July import group, sampled within a few weeks of their arrival. Stress factors involved in importing include confinement in quarantine, a change in ration, international and national transport, and acclimatisation to a new environment, management system and peer group in New Zealand. This assessment found a significant difference in the prevalence of the two risk factors, identified at the individual animal level, across the five farms. The proportion of cria in the flock ranged between 4.3% and 26.5% (p<0.001), while the proportion of alpacas exposed to the May imports ranged between 0% and 92.6% (p<0.001). The worst affected farm (Farm 2) had the highest proportion of cria and also the highest proportion of individuals exposed to the May import consignment. Investigators identified a number of management differences (farm-level risk factors) that are likely to influence prevalence and that may have affected the relationships identified at the individual animal level. These include variability of faecal pick-up by the farmer, differences in stocking density, paddock management that encourages midden formation, grazing system (Figure 3) and the numbers of recently imported animals. A further factor that Figure 1: Section of ileum showing multifocal ulceration, reduced crypt formation, neutrophil, macrophage and plasma cells infiltrates in the muscularis mucosa and submucosa, and large numbers of various life stages of Eimeria macusaniensis in the lamina propria (x40) Figure 2: Section of ileum showing numerous developing stages of Eimeria macusaniensis in the lamina propria, including meront (M) and thick-walled oocysts (O) (x400) would affect the resident animals’ exposure is the variability of quarantine period imposed on new arrivals. For alpaca imports, this separation traditionally serves the purpose of ensuring more intensive management of the imports as they recover from the stresses of importation but also importantly serves to protect resident stock from introduced diseases or parasites. The severely affected animal described in the case report appears typical of sporadic cases reported from Australia(4), the USA (Cebra C, personal communication) and the United Kingdom(13). Eimeria macusaniensis infestation predominates in the ileum with dramatic parasite numbers obliterating normal intestinal architecture. Secondary changes include necrotising and/or bacterial enteritis, and multifocal ulceration. Most pathology occurs in the small intestine, leading to weight loss and hypoproteinaemia. As a result sick alpacas may present with acute weakness or sudden death. Diarrhoea is not a frequent finding (Jones J, personal communication)(4). During the investigation it became apparent that this was not the first incursion of E macusaniensis. Evidence included the presence of E macusaniensis in alpacas not exposed to the May consignment on two of the four affected farms, a retrospective Australian quarantine report of the coccidian in alpacas destined for New Zealand in April 2004 (a finding identified at a review of Australian quarantine test results, requested by MAF as part of this investigation), and the detection of E macusaniensis in an alpaca on a fifth farm that was not associated with the others. New Zealand Import Health Standards have never required testing and/or prophylactic treatment for this parasite. Since this organism did not appear to be a recent introduction to New Zealand, and as its pathogenicity may have been overstated in the literature, MAF removed interim containment measures on the affected farms. The findings of this investigation are a reminder that import testing protocols do not exclude all diseases and pests, that farmers should remain vigilant and take measures to protect resident stock and report anything unusual to MAF through the 0800 809 966 exotic disease and pest freephone. Acknowledgements The authors would like to thank all Gribbles Veterinary Pathology staff, veterinarians, AgriQuality Limited field personnel, and Biosecurity New Zealand’s Pre Clearance team, who contributed during the initial investigation and follow-up survey. The invaluable input of Marsha Stevens and Rob Fairley (Gribbles Veterinary Pathology Christchurch), Melanie Taylor and John Gill (Gribbles Veterinary Pathology Invermay) and Donald Arthur (Selwyn Rakaia Veterinary Services) is especially acknowledged. Alastair Johnstone (Institute of Veterinary, Animal and Biomedical Sciences, Massey University) is also acknowledged for providing photographs of the histology sections. References (1) Guerrero C. Coccidia (Protozoa: Eimeriidae) of Alpaca Lama pacos. Journal of Protozoology 14, 613-6, 1967. (2) Guerrero C, Hernandez J, Bazalar H, Alva J. Eimeria macusaniensis (Protozoa: Eimeriidae) of the alpaca Lama pacos. Journal of Protozoology 18, 162-3. 1971. (3) McKenna PB. Some new host-parasite records. Surveillance 28(1), 4-5, 2001. (4) Lenghaus C, O’Callaghan MG, Rogers C. Coccidiosis and sudden death in an adult alpaca (Lama pacos). Australian Veterinary Journal 82, 711-2, 2004. (5) Rosadio RH, Ameghino EF. Coccidial infections in neonatal Peruvian alpacas. Veterinary Record 135, 459-60, 1994. (6) McKenna PB. Eimeria macusaniensis in camelids - a brief review. Surveillance 33(4), 8-10, 2006. (7) Jarvinen JA. Prevalence of Eimeria macusaniensis (Apicomplexa: Eimeriidae) in midwestern Lama spp. Journal of Parasitology 85, 373-6, 1999. (8) Rohbeck S, Gauly M, Bauer C. On the biology of Eimeria macusaniensis, an intestinal parasite of South American camelids. 19th International Conference of the World Association for the Advancement of Veterinary Parasitology. New Orleans, USA, 2003. (9) Rickard LG, Bishop JK. Prevalence of Eimeria spp. (Apicomplexa: Eimeriidae) in Oregon llamas. Journal of Protozoology 35, 335-6, 1988. (10) Schrey CF, Abbott TA, Stewart VA, Marquardt WC. Coccidia of the llama, Lama glama, in Colorado and Wyoming. Veterinary Parasitology 40, 21-8, 1991. (11) Leguia G. The epidemiology and economic impact of llama parasites. Parasitology Today 7, 54-6, 1991. (12) Rickard L. Update on Llama medicine: Parasites. Veterinary Clinics of North America: Food Animal Practice 10, 239-47, 1994. (13) Chief Veterinary Officer, U.K. The Report of the Chief Veterinary Officer - Animal Health 2004. 2004. Thomas Rawdon Andrew McFadden Caleb King Investigation and Diagnostic Centre (Wallaceville) Biosecurity New Zealand PO Box 40 742 Upper Hutt Email: Thomas.Rawdon@maf.govt.nz Verona Mitchell Mosgiel Veterinary Services Limited 4 Cargill Street Mosgiel Mark Howell Surveillance and Incursion Response Biosecurity New Zealand Wellington
I would like it noted, that all alpacas are checked in quarantine for all parasites, and coccidia, liver fluke and salmonella.

All counts are also taken if any are found., and treatment is required and retested to confirm any alpaca in quarantine destined for export to NZ, will be zero for all tests.
These tests until death of Paul Presedente, were tested by Paul himself, and since by technicians taught by Paul.
These tests are all completed by Australian GOvernment Laboratories, and all alpacas are zero for EMAC, and any other coccidia, as well as Liver Fluke, Salmonella (tested twice), and stomach parasites, as well as mites, and ectoparasites.

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