Annex

1. SOURCE FREEDOM of megalocytiviruses for gouramis, bettas, paradise fish, cichlids and poeciliids

The certification of (2A) ‘The fish originate from a country, zone or compartment determined by the Competent Authority to be free from Megalocytivirus based on the procedures described in the Department of Agriculture and Water Resources Import Condition’ must be based on active targeted surveillance in the country, zone or compartment described under Sections 1.1 to 1.9.

1.1. Surveillance must demonstrate the absence of megalocytiviruses and suspected clinical signs in the source population during a surveillance period specified by the Competent Authority. The active targeted surveillance program must include a minimum of two rounds of sampling and testing for megalocytiviruses in the source population of fish to be exported to Australia. Chapter 1.4 Aquatic Animal Health Surveillance of the OIE Aquatic Animal Health Code (OIE Code) should be referred to when designing a surveillance program for health certification purposes.

1.2. The Competent Authority may recognise a zone or compartment as free of megalocytiviruses based on negative test results using internationally recognised molecular diagnostic protocols referred to under Section 1.6 below. The testing regimens apply in addition to the requirement for an absence of suspected clinical signs associated with megalocytiviruses, and that basic biosecurity conditions, as defined in Section 1.8, must have been continuously met during the surveillance period.

Sample size for source freedom

1.3. The sample size within a source population should be calculated using the guidelines in Chapter 1.4 Aquatic Animal Health Surveillance of the OIE Code. Source populations of fish to be included in a sampling regime may be of a single susceptible species if epidemiologically isolated, or if mixed populations of susceptible species, then these may be sampled using a stratified approach as described in the aforementioned chapter. Suggested sampling methodology is provided below for situations where disease prevalence in the source population is unknown (Section 1.4) or known (Section 1.5).

1.4. Should the health status of source fish be unknown then the sampling design of targeted active surveillance for demonstrating megalocytivirus-free status of a country, zone or compartment must require a minimum level of sampling appropriate for a 5 % assumed prevalence with a 95% confidence level of detecting megalocytiviruses in a source population. Examples of sample sizes for various combinations of design prevalence, diagnostic sensitivity and specificity values are provided in Table 1.

1.5. Should the Competent Authority be aware of the likely megalocytivirus prevalence in the source population (based on the previous baseline surveillance and/or published scientific literature), the sampling size can be designed with an expected prevalence that differs to 5 %. The sampling design should take into account epidemiological factors, including but not limited to, the virus characteristics (e.g. transmissibility, incubation period, the virus infecting homogenously or clustering within the population), population dynamics and biosecurity practices (e.g. the number of ponds/aquaria, physical structures of the farm, frequency and duration of population mixing, water intake and water sharing).

Diagnostic tests

1.6. The appropriate test methods for the purpose of demonstrating freedom of megalocytiviruses are those based on the polymerase chain reaction (PCR). These molecular tests must be capable of detecting subclinical carriers of the virus, such as two-step (nested) PCR and quantitative PCR methods published in peer-reviewed journals or equivalent. The standard PCR test for red seabream iridovirus (RSIV) described in the current OIE Manual of Diagnostic Tests for Aquatic Animals (OIE Manual) is not considered suitable at present as there is insufficient evidence that the test is capable of detecting the presence of megalocytiviruses in subclinically infected fish.

Ongoing surveillance

1.7. Ongoing surveillance to demonstrate disease freedom should involve twice yearly sampling and testing at a level appropriate for a 10 % assumed prevalence with a 95% confidence level of detecting megalocytiviruses in a source population. The conditions for ongoing surveillance to demonstrate disease freedom will only be recognised following two years of continuous active targeted surveillance with no positive detections of megalocytiviruses, an absence of suspected clinical signs and demonstration that basic biosecurity conditions (as defined Section 1.8) were continuously met for the period.

1.8. Basic biosecurity conditions are defined in the OIE Code as a set of conditions applying to a particular disease, and a particular compartment, zone or country, required to ensure adequate disease security, such as:

1.8.1. the disease, including suspicion of the disease, is compulsorily notifiable to the Competent Authority; and

1.8.2. an early detection system is in place within the compartment, zone or country; and

1.8.3. import requirements to prevent the introduction of disease into the compartment, zone or country, as outlined in the OIE Code, are in place.

Health certification

1.9. Exporting countries are required to provide health certification attesting to freedom from megalocytiviruses for all fish destined for export to Australia. This includes attestations of disease freedom for fish that may have been sourced from a different country to the final export country. Attestations of disease freedom in these circumstances must be based on satisfactory certification arrangements between the original source country and the final exporting country.

2. BATCH TEST NEGATIVE for megalocytiviruses for gouramis, bettas, paradise fish, cichlids and poeciliids

The certification of (2B) ‘The fish originate from a batch that has been tested by the Competent Authority and found negative for Megalocytivirus using a testing method that is consistent with the Department of Agriculture Import Condition’ must be based on batch testing described under Sections 2.1 to 2.6.

2.1. Pre-export batch testing for megalocytiviruses is not required if fish are sourced from a country, zone or compartment recognised by the Competent Authority of the exporting country to be free of the virus on the basis of a surveillance program consistent with Section 1 above.

Batch definition

2.2. For the purposes of batch testing for freedom from megalocytiviruses, a batch can be defined as fish of susceptible species sharing water in a single holding system at the time of sample collection that have remained epidemiologically isolated for at least 14 days from fish not of equivalent health status prior to export.

Diagnostic tests

2.3. Refer to the information in Section 1.6.

Sample size for batch testing

2.4. The sample size within a batch should be calculated using the guidelines in Chapter 1.4 Aquatic Animal Health Surveillance of the OIE Code. The design of a pre-export batch testing surveillance program for demonstrating freedom from megalocytiviruses must require a minimum level of sampling appropriate for a 5 % assumed prevalence. Examples of sample sizes for various combinations of design prevalence, diagnostic sensitivity and specificity values are provided in Table 1.

2.5. For some species, the number of animals constituting a batch is not large enough to meet the required sample size for pre-export batch testing. The Competent Authority of the exporting country may make an alternative arrangement for the suitable sample size for batch testing, either generally or case-by-case. Such arrangements should be supported by scientific data that would clearly demonstrate equivalence of the alternative arrangement.

2.6. All consignments of gouramis, bettas, paradise fish, cichlids and poeciliids must be held in quarantine at the export premises while being sampled for testing and remain under biosecurity control at the export premises until the results of the tests are available. Only fish from batches that return negative results are permitted to be exported to Australia.

3. Health certification for imported fish

Exporting countries are required to provide health certification attesting to freedom from megalocytiviruses for all fish destined for export to Australia. This includes attestations of disease freedom for fish that may have been sourced from a different country to the final export country. Attestations of disease freedom in these circumstances must be based on satisfactory certification arrangements between the original source country and the final exporting country.

Table 1: Examples of sample sizes generated by FreeCalc (Sergeant 2015) for various combinations of design prevalence, test sensitivity and specificity values (http://epitools.ausvet.com.au/content.php?page=FreeCalc2).