Pilot field trial of the EG95 vaccine against ovine cystic echinococcosis in Rio Negro, Argentina: 8 years of work

Accepted Manuscript Title: Pilot field trial of the EG95 vaccine against ovine cystic echinococcosis in Rio Negro, Argentina: 8 years of work Authors: Edmundo Larrieu, Guillermo Mujica, Daniel Araya, Jose Luis Labanchi, Marcos Arezo, Eduardo Herrero, Graciela Santill´an, Katherina Vizcaychipi, Leonardo Uchiumi, Juan Carlos Salvitti, Claudia Grizmado, Arnoldo Calabro, Gabriel Talmon, Luis Sepulveda, Jose Maria Galvan, Marta Cabrera, Marcos Seleiman, Pablo Crowley, Graciela Cespedes, Mariela Garc´ıa Cachau, Lilia Gino, Leonardo Molina, Jose Daffner, Charles G. Gauci, Meritxell Donadeu, Marshall W. Lightowlers PII: DOI: Reference:

S0001-706X(18)30956-2 https://doi.org/10.1016/j.actatropica.2018.12.025 ACTROP 4870

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Acta Tropica

Received date: Revised date: Accepted date:

6 August 2018 15 December 2018 17 December 2018

Please cite this article as: Larrieu E, Mujica G, Araya D, Labanchi JL, Arezo M, Herrero E, Santill´an G, Vizcaychipi K, Uchiumi L, Salvitti JC, Grizmado C, Calabro A, Talmon G, Sepulveda L, Galvan JM, Cabrera M, Seleiman M, Crowley P, Cespedes G, Garc´ıa Cachau M, Gino L, Molina L, Daffner J, Gauci CG, Donadeu M, Lightowlers MW, Pilot field trial of the EG95 vaccine against ovine cystic echinococcosis in Rio Negro, Argentina: 8 years of work, Acta Tropica (2018), https://doi.org/10.1016/j.actatropica.2018.12.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Pilot field trial of the EG95 vaccine against ovine cystic echinococcosis in Rio Negro, Argentina: 8 years of work Edmundo Larrieua,c,*, Guillermo Mujicab, Daniel Arayab, Jose Luis Labanchib,c, Marcos Arezob,c, Eduardo Herrerob, Graciela Santillánd, Katherina Vizcaychipid, Leonardo Uchiumib, Juan Carlos Salvittib, Claudia Grizmadob, Arnoldo Calabrob, Gabriel

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Talmonb, Luis Sepulvedab, Jose Maria Galvanb, Marta Cabrerad , Marcos Seleimanb,

Pablo Crowleyc, Graciela Cespedesd, Mariela García Cachaua, Lilia Ginoa, Leonardo Molinaa, Jose Daffnerc, Charles G. Gaucie, Meritxell Donadeue, Marshall W.

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Lightowlerse

a Facultad de Ciencias Veterinarias, Universidad Nacional de La Pampa, General Pico,

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Argentina

b Ministerio de Salud, Provincia de Río Negro, Viedma, Argentina

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c Escuela de Veterinaria, Universidad Nacional de Rio Negro, Choele Choel, Argentina

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d Instituto Nacional de Microbiología “ANLIS-MALBRAN”, Buenos Aires, Argentina

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Victoria 3030, Australia

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e University of Melbourne, Veterinary Clinical Centre, 250 Princes Highway, Werribee,

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GRAPHICAL ABSTRACT

Pilot field trial of the EG95 vaccine against ovine cystic

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echinococcosis in Rio Negro, Argentina: 8 years of work

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Pilot field trial of the EG95 vaccine against ovine cystic echinococcosis in Rio Negro, Argentina: second study of impact Vaccination of potential intermediate hosts of E. granulosus with the EG95 vaccine could be used to reduce E. granulosus transmission

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Was observed a statistically significant reduction in the prevalence of ovine CE, in the cystic number and in the in the numbers of farms with infected sheep

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The arecoline test did not show statistically significant differences in the infection prevalence in dogs but with coproELISA the different was statistically significant between the initial and impact studies

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Vaccination was effective even in a difficult, remote environment where only approximately half the lambs born into the communities were fully vaccinated

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ABSTRACT

Cystic echinococcosis (CE) is endemic in the Rio Negro province of Argentina. After 30

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years of control using praziquantel in dogs the transmission rate to humans and sheep has decreased significantly, however transmission persists. The objective of the study was to

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assess the inclusion of the EG95 for sheep in the control program and to determine the vaccine's operative feasibility in field conditions. An intervention study was defined in

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Rio Negro Province in Argentina comprising, in total, an area of 5820 Km2. Lambs received two vaccinations with the EG95 vaccine followed by a single booster injection

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when the animals were 1-1.5 years of age. Vaccination of lambs born into one trial site was introduced and continued for 8 years. Evidence for Echinococcus granulosus transmission was monitored before and after vaccination by coproantigen ELISA in faecal samples of dog, purgation of dogs to detect E. granulosus worms, necropsy on

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adult sheep and by ultrasound screening in children of 6 to 14 years old. 29323 doses of vaccine were applied between 2009 and 2017, which a vaccination coverage of 80.1%/85.7% (57.3% average for fully vaccinated). Before the introduction of the vaccine 56.3% of the 6-year-old sheep were infected with E. granulosus at necropsy and 84.2% of the farms had infected sheep; 4.3% of the dogs were positive for E. granulosus infection using the arecoline test, and with coproELISA 9.6% of dog fecal samples were 3

positive and 20.3% of the farms had infected dog.After the vaccine was introduced, 21.6% of sheep older than 6 years were found to be infected at necropsy and 20.2% of the farms were found to be infected; in dogs, 4.5% were found positive for E. granulosus using arecoline purgation and with coproELISA 3.7% of samples were positive, with 8.9% of farms having a positive dog . In 2016 only one case of E. granulosus infection was diagnosed by US screening in a 6-14 years old child. Included in the analysis are discussions of difficulties experienced in the field which affected correct vaccine

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administration as well as social features and practices that may impact on echinococcosis

control and the EG95 vaccination program in Rio Negro. Vaccination of sheep with the

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EG95 vaccine provides a valuable new tool which improves the effectiveness of CE

control activities. Vaccination was effective even in a difficult, remote environment where only approximately half the lambs born into the communities were fully

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vaccinated.

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Keywords: Cystic echinococcosis, EG95, Vaccination, Diagnosis, Control, Sheep

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∗ Corresponding author at: Ministerio de Salud, Laprida 240, 8500 Viedma, Argentina. Tel.: +54 2920 430007; fax: +54 2920 430007. E-mail addresses:

Introduction

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1.

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[email protected] (E. Larrieu)

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Echinococcosis (in its cystic, alveolar and polycystic forms) is a group of zoonoses caused by the larval forms of parasitic cestodes of the genus Echinococcus. Echinococcus granulosus is the etiological agent of cystic echinococcosis (CE), which from a taxonomic perspective is currently considered a multi-species

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complex referred to as E. granulosus sensu lato (s.l.). This complex includes the species E. granulosus sensu stricto (ss), genotypes G1 / G2 / G3. E. granulosus s.s. (particularly genotype G1) is the species with the highest prevalence and widest global distribution and is responsible for approximately 80% of human cases of CE globally (Alvarez Rojas et al., 2014), including in South America (Cucher et al., 2016).

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The predominant strain of E. granulosus causing both human and livestock infections in Rio Negro is the G1 strain (Rozensvit et al., 1999; Cutcher et al., 2016). CE may cause morbidity, disability and death in humans. It persists in nature through a cycle comprising the two lifecycle stages of the parasite. The adult form parasitizes exclusively canids and, in general, does not generate any pathology. The larvae or hydatids are located in the organs and tissues of herbivores and omnivores (sheep, cattle, swine, equines, South American camelids, goats), causing decrease in the your

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productivity (Eckert et al., 2002).

In the Province of Rio Negro, Argentina, the slaughter of adult sheep and goats

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for family consumption, and the viscera being used for feeding the dog, completes the parasite’s lifecycle, hence the practice of home slaughter of small animals is the main risk factor for the spread of the disease. Slaughterhouses of small towns with poor hygiene conditions that allow the release of raw viscera out of the establishment, and the death of

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infected intermediate hosts in the field, are other mechanisms that contribute to the

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transmission cycle.

Infection in humans occurs after accidental ingestion of the parasite eggs from the

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feces of an infected dog through contaminated water, food, or by direct contact with

economy (Guarnera et al., 2013).

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parasitized dogs. As a consequence, CE impacts public health, animal health and the rural

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The CE control program in the Province of Río Negro was launched in 1980, covering 120013Km2 of 7 provincial departments in two stages (4 departments began the

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deworming of dogs in 1980 and another 3 in 1985). The actions, from the beginning to the present, are mainly supported by the infrastructure of primary health care. Thus, health workers carried out the deworming of 11500 dogs with praziquantel, four times per year

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during home visits.

They gave the praziquantel pills to the farmers to dose their own dogs.

Veterinarians of the control program were responsible for the surveillance of CE (Perez

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et al., 2006), initially by arecoline test to identify infection in dogs, and from 2003, by coproELISA test to identify infected dogs and hence sheep farms with active transmission. Both the community health workers and control program veterinarians were also responsible for health education strategies aimed at schoolchildren and rural people. The surveillance was based on screening methods. From the beginning of the program, surveys were conducted for the early detection of CE in people, especially in 5

schoolchildren from 6 to 14 years of age, initially through serology (double diffusion 5, ELISA) associated with early surgical treatment, and, since 1997, ultrasonography (US) combined with a treatment scheme based on albendazole. In 1984/6 the first surveys using US in children aged 6-14 years of age showed a prevalence of 5.6%. and in 2015 the prevalence rate in US surveys of children aged 6 to 14 years of age reduced to 0.12% in the whole Rio Negro province. Thus, CE remains endemic, with stable rates of infection in dogs being significantly lower than at the

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beginning of the program and with a significant reduction in transmission to humans (Perez et al., 2006; Larrieu et al., 2012;).

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But, in some of the regions included in the program (for example Rio Chico abajo,

Mamuel Choique and Anecon Gande), which present ideal ecological conditions for the sustainability of the E. granulosus lifecycle (Arezo, 2016), together with marginal social conditions, aggregation of small productive units and deficiencies in the sanitary

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infrastructure, usually in reservations of native population, the occurrence of new cases

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in children and adults is maintained (between 1995 and 2015, before vaccination, 38 new cases were identified, being the cumulative incidence rate 7800 per 100000 inhabitants).

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Based on this information, in 2009, vaccination of lambs with EG95 was

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incorporated in these regions (Larrieu et al., 2013; 2015). Vaccination of potential intermediate hosts of E. granulosus with the EG95

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recombinant vaccine could potentially be used to reduce the level of E. granulosus transmission. Several experimental tests have been carried out in Argentina, China and

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Australia showing that it produces high levels of immunity (Heath et al., 2003; Lightowlers et al.,1999).

The objective of this study is to update and extend the evaluation of the results of

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the vaccination program with EG95 carried out for 8 years, including the evaluation of the transmission to the dog, and to provide an analysis of the conditions and limiting

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factors affecting a CE control program incorporating vaccination against CE in sheep.

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Materials and methods

2.1. Study area

The study region consisted of the areas of Rio Chico Abajo (Department Ñorquinco), Anecón Grande (Department Pilcaniyueu), Mamuel Choique (Department 6

25 de Mayo) and Nahuel Pan (Department Bariloche), located in the Province of Rio Negro, Patagonia region, Argentina (Table 1, Figure 1). In 2009, there were 79 farmers with a total of 8483 sheep, the majority being in Rio Chico, all of them small farmers (average 107 sheep per producer). All the study areas are Mapuche native communities, including 2 of them (Anecon Grande and Nahuel Pan) organized in a reserve form, with a Lonco (cacique), common property land and without subdivisions of the land through fencing.

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The climate and the lack of road infrastructure limit the accessibility to the farms

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during much of the autumn and winter.

Farm management practices and other factors affecting CE

There are some production characteristics that vary between the vaccination areas. Many farmers of Rio Chico Abajo have their properties alongside the river which allows

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them to have good quality pastures year-round. These pastures allow them in some cases,

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the farmers to buy from dry areas old sheep that are near the end of their productive life and to get from them one further shearing and a lamb. These animals are purchased in

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areas outside the vaccination zone. In Nahuel Pan, likewise, there is no organized animal

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management, sheep and rams live together all year round so at the time vaccinations were undertaken (December-February) lambs ranged from 15 days to 4/5 months of age.

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Between 2009 to 2011 the region was affected by several years of drought as well as the eruption of the Puyehue volcano in Chile that left a layer of volcanic ash in the

of farmers.

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area, generating an animal mortality close to 50% and an abrupt decrease in the number

In all the study sites there is a Health Centre with a health worker (except Nahuel

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Pan actually), supported by a periodic medical visit. In the study area there are operational differences between the health workers. For

example, in Rio Chico and Mamuel Choique, the health worker carries out home visits to

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distribute antiparasitic drugs four times per year, deworming the dogs and ensuring the ingestion of the drugs by the dogs combining it with minced meat. In Anecon Grande, the health worker travels by horse and leaves the antiparasitic drug with the dog owners, who are responsible for the deworming. In Nahuel Pan there hasn’t been a health worker in recent years, and residents must go to the Health Centre to obtain the antiparasitic drugs. In Rio Chico area there is a special epidemiological situation, since sometimes old sheep

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can be brought from non-vaccination areas, due to the availability of better pastures in the areas near the river.

2.2. Vaccination of sheep The EG95 sheep vaccine used throughout the study was prepared and donated by the University of Melbourne, lyophilized and bottled in 50 or 100 dose vials together with an adjuvant. The vaccine was reconstituted with distilled water and applied

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subcutaneously in the neck, at a dose of 50 µg of EG95 protein in a volume of 2 ml (Larrieu et al., 2015)

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The vaccination scheme was designed to achieve a potential 100% immunity (3

doses over approximately 12 months) and to minimize the personnel and logistic requirements for its application, as well as the number of doses needed (considering the potential need to buy the vaccine in the future). While many of the farms also kept goats,

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initial observation of the low prevalence of echinococcosis in goats (unpublished), there

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being little local habit of the slaughter of adult goats, and due to the difficulties working with livestock in the region, goats were excluded from the vaccination program.

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The 3 doses of vaccine were applied to lambs. The first dose at 30 days of age, the

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second at 60 days of age before weaning and a third booster dose at 1-1.5 year of age, applied at the same time when the lambs of the following year received their second dose.

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In this way the vaccination teams, made up of veterinary personnel of the control program, attended the study sites to vaccinate for one week every December and January

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starting in 2009. Arrangements so the farmers had their herds corralled on the designated vaccination day were made through previous home visits by the health worker and notification through National AM radio. All the vaccinated animals were identified with

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an ear tag, using a different colour tag for each year. Therefore, each animal with an ear tag had at least 1 dose of vaccine, but the animals were not tagged with individual identifying numbers (Larrieu et al., 2015). At each visit all lambs that were present were

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vaccinated. Those that the producer did not manage to enclose or escaped from the corrals during the time that the vaccinations were being carried out failed to be vaccinated. In these and other areas of the Rio Negro Province, the regular control activities involving treatment of dogs with praziquantel continued.

2.3. Assessment methods

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Figure 2 shows the timeline of the activities developed, from the initial diagnosis until the impact study: Before the start of the vaccination, in 2009, a baseline of infection was established in the different hosts, using indirect methods: CoproELISA as a screening method with confirmation by Western Blot (WB) in faecal samples of dog obtained from the environment, being the observation units a) positive samples to coproELISA and b) farms with at least one positive sample to coproELISA (Guarnera et al., 2000; Cavagion et al.,

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2005), and using ELISA as a screening test on sera from sheep with confirmation by WB (Gatti et al., 2007).

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Direct methods were also used for visualization of the parasite: necropsies in adult sheep with diagnostic confirmation by histology (Cabrera et al., 2003), being the observation units a) positive sheep at necropsy and b) farms with at least one positive

sheep at necropsy and arecoline test in dogs (unit observation, dog) (Perez et al., 2006).

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The same strategy was conducted at the end of the study period (necropsy in 2015 in

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sheep and arecoline test and coproELISA in dogs in 2017, when it was anticipated that 100% of the flock in the area in which the vaccine had been applied would have comprised

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only vaccinated animals (assuming 100% coverage of the animals) with the exception of

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those brought unvaccinated from elsewhere.

The selection of the farms to be evaluated was carried out through simple

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randomized and statistically representative sampling with a 95% confidence and a 20% margin of error, with the exception of the arecoline test that was applied in high dog

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population areas, with voluntary attendance by their owners (not randomized). Specific screening in children of the school of the vaccination area was made in 2016 (Perez et al., 2006; Larrieu et al., 2011).

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The statistical analysis was conducted with EPIDAT 3.1, estimating proportions

and their 95% confidence intervals, Chi square of association or Fisher Exact Test with a level of significance of p <0.05 for each of the analytical techniques used to compare

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prevalence of the infection before vaccination and at the end of the study period, and Chi square of Pearson with a level of significance of p <0.05 to compare the prevalence between areas.

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Results

29323 doses of vaccine were applied between December 2009 and January 2017, 17894 in Rio Chico, 1056 in Nahuel Pan, 2220 in Mamuel Choique and 8153 in Anecon Grande; which represents a vaccination coverage of 83.5% at the first dose, 80.1% in the second dose and 85.7% at the third dose (83.5x80.1x85.7 = 57.3% average for fully vaccinated across the area). Coverage is estimated as the proportion of lambs vaccinated during the study period in relation to the lambs present in the same period (Table 2 and 3).

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Evaluation with ELISA/WB in two years old sheep: An initial assessment was

made in 2011 of the impact of the vaccine on infection levels in 238 two years old sheep

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being the differences between base and impact study significative (p < 0.001) (Larrieu et al., 2013).

Evaluation with necropsy in old sheep: Before the introduction of the vaccine, in 2009, 56.3% of the 6-year-old animals were positive at necropsy whereas the impact

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studies also based on adult sheep necropsies that were undertaken in 2015 showed the

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prevalence had decreased to 21.1% 5 years after the use of the vaccine (p value= 0.03). The number of cysts per animal decreased from 1.4 to 0.3. All cysts were small (<1 cm)

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(Larrieu et al; 2015). In relation to the number of farms with infected sheep there was a

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significant difference (p value= 0.002) between the level at baseline and the study of impact. Comparisons between the different areas in which the control activities were

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being undertaken did not reveal any differences in the proportion of farms with infected sheep (P<0.05; Table 4).

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Evaluation with arecoline test in dogs: Between 2009/2017, the arecoline test did not show statistically significant differences in the infection prevalence in dogs determined using arecoline treatment between the initial and impact studies (4.5% to

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4.3%, p value= 0.8), while among the different work areas there were statistically significant differences observed in the baseline (p value= 0.006) but not in the impact study (p value= 0.7).

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Evaluation with coproELISA in dogs and farms: using the coproELISA for

diagnosis of infection in dogs, the differences were statistically significant (p value= 0.04) between baseline (2009) and impact (2017) studies in the proportion of positive samples; no differences were evident between the different areas in which the control work was being undertaken (p value > 0.05). In relation to the farms, the differences were not significant among producers with farms with active transmission (by the presence of an

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infected dog) (p value= 0.1); no statistically significant differences were evident between the baseline and impact evaluation across the different work areas (p value > 0.05). Evaluation with US: Only 1 asymptomatic case was identified in the screening in 2016 in 84 schoolchildren of 6 to 14 years old in school of work area (1.1%) being a lung cyst of 10 cm. No symptomatic case was diagnosed in the period.

Discussion

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4.

Torgerson (2003) developed a mathematical model to simulate the impact of

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different CE control options in circumstances where transmission was occurring between

domestic dogs and sheep. The effectiveness of different interventions, or intervention combinations, was affected by the initial infection pressure in dogs and sheep prior to the initiation of control measures, and the proportion of dogs and sheep that were treated. A

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combination of anthelmintic treatment of dogs and vaccination of sheep involving at least

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a 75% compliance rate achieved a relatively rapid decline in E. granulosus transmission irrespective of the initial level of transmission. The effectiveness of this intervention

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scenario was enhanced where an education component led to a 50% reduction in feeding

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of offal to dogs, such that control could be achieved in 3-5 years. In the present study, sheep were vaccinated at a coverage close to 85% for each

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dose. Full vaccination was considered to involve two immunizations in lambs and one additional vaccination when the animals were approximately 1 year of age. This being

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the case, three immunizations at a compliance rate of 80%/85% for each vaccination represents an effective vaccination compliance of 57.3%. Praziquantel treatment of dogs was specified on 4 occasions per year (effectively a 3 monthly treatment), although the

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coverage probably does not reach 75% in each round. With an initial prevalence of infection in sheep representing an extreme endemicity (almost 50%), but a proportionally low prevalence of infection in dogs (around 5%), the assumptions of the model seem to

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be fulfilled with a prevalence of infection in sheep at 7 years close to 20%, which allows maintenance of the transmission cycle between insufficiently vaccinated sheep and dogs that are insufficiently dewormed, but with expectations to sustain a trend towards effective control after 15 years of activities. The impact studies which were based on necropsy results on adult sheep (Larrieu et al., 2015) showed that a pronounced decrease of the prevalence, number of cysts per sheep and number of producers with infected sheep (56.3% to 21.1%, 1.4 to 0.3 y 84.2% 11

to 22.2% respectively, all with significant difference). However, the analysis comparing the results according to the geographical and production characteristics, land tenure and the strategy of the health workers, among the 4 areas, is inconsistent with the different prevalence rates resulting in no significant differences except in the baseline study using the arecoline test. The introduction of non-vaccinated adult sheep into the Rio Chico area introduced a disturbing element in the program and may have reduced the effects of vaccination in

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reducing CE prevalence in dog.

The dogs from the study which were positive in the arecoline test correspond to 1

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dog of a farmer in Rio Chico (usually with dogs dewormed by the health worker, probably more than 60 days after its last deworming and with access to sheep slaughtered at home, from a non-vaccination zone), 2 dogs in Nahuel Pan (area without a health worker to deworm the dogs, dogs that can roam outside the deworming zone and farmers that lack

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sheep management making timely vaccination difficult) and 2 dog in Anecon Grande

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(one of farmer with goats only). Infection in dogs detected with the arecoline test, appears to have been due to risk factors associated with the animals having access to viscera from

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livestock that were not involved in the vaccination program and/or the ability of the dogs

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to roam and feed outside of the vaccination area.

Goats were not included in the vaccination program for reasons of time,

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operability and assuming the low epidemiological impact of echinococcosis in goats in the area. One dog found to be infected with E. granulosus came from a property where

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the owner kept only goats, however he does purchase sheep for home slaughter from neighbours and hence the source of the infection in that dog is unclear. Anecdotal evidence available to the authors suggests that CE is uncommon in goats in the areas in

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which the intervention was undertaken, however recent and definitive data about CE in goats is not available. Potentially transmission through goats may have contributed to the continuing presence of E. granulosus infections detected in dogs despite a dramatic

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reduction in CE in the sheep population. As a result of this evaluation, the decision not to vaccinate goats is subject to further analysis and consideration. A future study will elucidate the possible role of goats in maintaining the infection in dogs. The outcomes of this project will determine whether the decision to exclude goats was appropriate or not. Serological monitoring of the immune response to vaccination conducted in relation to the present study (Larrieu et al., 2017) found that after the third dose with the EG95 vaccine at one year of age, the specific IgG responses detected in the serum of 12

sheep increased to a level higher than that observed after the second dose, and that this response was maintained over time, for at least 5 years, which would be sufficient to avoid infection of the sheep and/or development of fertile cysts during the period of its useful life in the region. This highlights the importance of the sheep receiving the full 3-dose vaccination regime over their first year of life. The effectiveness of the vaccination program was adversely affected by our inability to deliver all 3 doses to approximately 40% of the sheep due to the inherent difficulties of working in remote communities with

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poor animals handling facilities and poor methods of communication between the control staff and the residents (public radio).

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The only child with a diagnosis of CE was in a child under 15 years of age. This

patient was identified with a 10 cm cyst in the lung. It is possible that the infection may have been acquired before or close to the start of the vaccination program. Although the time elapsed since the introduction of the vaccine is insufficient to evaluate a stop in the

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transmission to man, the regular deworming activities conducted since 1980 that are

downward trend in the occurrence of new cases.

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enhanced by vaccination, seem to be associated positively with maintaining the

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It is recognized that since the development of praziquantel at the beginning of the

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1980s as an antiparasitic agent against CE, there is an effective tool to control the disease. However, the successes achieved using dog treatment as the only major control tool have

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been limited in many areas of the world. This is likely to be associated with the difficulties in having the logistics and means to effectively deworm 100% of dogs with a frequency

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of 8 times per year, for more than 10 years in remote rural areas which are difficult to access (Larrieu and Zanini, 2012; Craig et al., 2017). Vaccination of sheep is an effective new tool, although it some of the same

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challenges as does the use of praziquantel, that is, the need to have logistics and means to vaccinate twice a year in remote rural areas. Although resources are only required twice a year, as opposed to 4 to 12 times per year in deworming programs, the number of

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animals involved is much higher in the case of vaccination. The combination of both dog treatments and livestock vaccination is predicted to

achieve better results in stopping transmission of the disease (Torgerson, 2003). The use of vaccination alone could be unwise because it would take many years until the total renewal of the sheep flock, and before observing the first results in dogs and people, which considering the experience with the use of praziquantel, would be unsustainable in terms of long-term financing for countries and regions where the disease is endemic. 13

Acknowledgements

Invaluable support for this work was provided by health workers Casimiro Prafil, Belen Montes, Bernardo Geraghty, Miguel Lopez, Eugenio Calfual and Gustavo Tartaglia. We thank

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them and their communities of Anecon Grande, Nahuel Pan, Mamuel Choique and Rio Chico

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review of species and genotypes of Echinococcus granulosus sensu lato in humans and natural domestic hosts. Trop,. Med. Int. Health. 21(2),166-75 Eckert, J., Gemmell, M.A., Meslin, F.X., Pawłowski, Z.S., 2002. Manual on Echinococcosis in Humans and Animals: A Public Health Problem of Global Concern. WHO/OIE, France, 272 pp Gatti, A., Alvarez, A.R., Araya, D., Mancini, S., Herrero, E., Santillan, G., Larrieu, E., Ovine

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immunoassay. Vet. Parasitol. 143, 112–121. Guarnera,

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la

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Heath, D.D., Jensen, O., Lightowlers, M.W. 2003. Progress in control of hydatidosis using vaccination-a review of formulation and delivery of the vaccine and recommendations for practical use in control programmes. Acta Trop 85,133–43.

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16

I N U SC R A M ED PT CC E A Figure 1. Work Area in Rio Negro Province, Argentina

17

N U SC R

I A

CC E

PT

ED

M

A

Figure2. Timeline. vaccination with EG95

18

N U SC R

I Average number

of

Area

Sheep

Number

Farmers*

sheep

of

A

Table 1. Details of the CE control study areas at the beginning of the vaccination program and after 8 years of vaccine use. Rio Negro, 2009/2017

M

sheep

Number

of

Dog

goats

Dog/

Health

Farmer

workers

Lat/Long

Km2

Farmers Km2

per

farmer

Rio

Chico

41/36

Nahuel Pan***

13/8

11/3

CC E

Mamuel

5012/2663

PT

Abajo**

2009/2017

ED

2009/2017

205/132

1306/205

2009/2017

2009/2017

2009/2017

2009/2017

122/74

6034/2899

173/143

4/4

2009/2017

1/1

13/11

16/17

0/75

48/36

4/4

1/0

A

79/58

100/68

2508/580

−41.9004

5

2.6/1.6

41/12

3/4

1/1

−41.7777

286

0.04/0.01

221

0,05/0,05

1054

0.07/0.05

−70.1369 1880/898

145/82

1324/370

46/30

3/3

1/1

−41.3215 −70.2742

8483/3898

107/67

9866/3934

*And 5 additional farmers that only have goats ** PZQ pills given to dogs 4x a year in meat by sanitary agent *** Farmers had to remove PZQ pills when they went to health centre.

19

0,07/0,06

−71.4932

Grande**

Total

542

−70.4761

Choique** Anecon

−41.7098

309/221

4/4

4/3

by

I N U SC R

Number lambs

of that

Abajo

Manuel

that

Coverage

Number of

Coverage

Total doses

(%)

lambs

(%)

applied

that

received

dose

second dose

third dose in

in 8 years

8 years

in

8039

8

88,1

6623

80,1

3432

85,6

18094

449

69,1

379

75,8

228

91,2

1056

1010

72,7

838

94,2

372

83,6

2220

3456

87,6

3287

92,8

1706

89,1

8449

CC E

Nahuel Pan

lambs

of

received

PT

Chico

(%)

Number

received first

years Rio

Coverage

ED

Area

M

A

Table 2. EG95 lamb vaccine doses applied and coverage achieved. Rio Negro, 2009/2017

Choique

A

Anecon Grande Total

29819

20

12954

83.5

11127

80.1

5738

85.7

29819

I N U SC R

A

Table 3. Number of animals vaccinated with EG95; Rio Negro Province, 2009-2017 First vaccination n (%V1)

Second vaccination n (%V1 )

Third vaccination n (%V1)

Proportion fully vaccinated %V2

Total vaccination

2009-2010

2725 (86.5)

2448 (77.8)

1308 (71.1)

47.8

6481

2010-2011

2138 (68.2)

1745 (55.5)

616 (96.2)

36.4

4499

2011-2012

1103 (95.9)

498 (43.3)

348 (92.2)

38.2

1949

2012-2013

1104 (92.4)

969 (81.0)

579 (89.4)

66.9

2652

1304 (88.0)

1017 (68.6)

680 (93.2)

56.2

3001

1460 (91.3)

1405 (87.8)

739 (86.9)

69.6

3604

ED

CC E

2014-2015

PT

2013-2014

M

Lamb Cohort

2015-2016

1733 (91,4)

1563 (82.3)

815 (88.6)

66.6

3975

2016=2017

1482 (78.1)

1314 (88.7)

862 (94.0)

65.1

3658

13049 (83.5)

10979 (80;1)

5947 (85.7)

57.3

29819

A

TOTAL

n: number of lamb vaccinated %V1: proportion of the animals available to be vaccinated that were actually vaccinated; %V2: proportion of animals receiving the full three-dose vaccination schedule

21

I N U SC R

Table 4. E. granulosus prevalence prior to (base) and following (impact) sheep vaccination. Rio Negro, 2009/2017 Area

Study

Farm with infected Dog

by Arecoline

necropsy**

Base

abajo

Impact

12 4 33.3

P value****

0.04

Nahuel Pan

Base Impact

PT

P value****

infections

CoproELISA

by Farm with infected dog by coproELISA***

Test***

n + %2 (CI95%)

n + %3 (CI95%)

n + % 4 (CI95%)

27 0 0.0

54 6 11.1

25 6 24.0

34 1 2.9

49 1 2.0

17 1 5.9

0.06

0.12

21 1 4.8

29 2 6.9

16 2 12.5

29 2 6.9

31 2 6.5

9 2 22.2

0.7

0.9

0.5

Base

8 6 75.0

25 0 0.0

16 1 6.3

9 1 11.1

Choique

Impact

3 0 0.0

20 0 0.0

20 0 0.0

5 0

0.25

0.4

CC E

Mamuel

0.02

Anecon

Base

5 5 100

16 3 18.8

26 3 11.5

Grande

Impact

3 0

33 2

36 2

P value****

0.004

0.16

Base

19 16 84.2 (60-97)

89

Impact

18

P value**** p1 value****

A

P value****

Total

1,4 2

6 5 83.3

ED

Rio Chico

M

n + %1 (CI95%)

by Dog

Test***

A

sheep

infections

0.0

6.1

5.6

0.0

9 3 33.1 14 1

7.1

0.34

0.1

125 12 9.6 (4-15)

59 12 20.3 (9-31)

116 5 4.3 (1-10)

136

45

0.0002

0.8

0.04

0.1

0.1/0.1

0.006/0.7

0.8/0.6

0.5/0.8

4 22.2 (6-47)

4 4.5 (1-11)

5 3.7 (1-8)

4

= total number of farms tested, += positive farms, %= percentage positive farms, CI95% confidence interval

= total number of dogs tested, += positive dogs, %= percentage positive dogs, CI95% confidence interval3

22

8.9 (2-21)

I N U SC R

3

= total number of faecal samples of dogs tested, += positive dogs samples, %= percentage positive, CI95% confidence interval

** 2009 base study, 2015 impact study. *** 2009 base study, 2017 impact study.

A

CC E

PT

ED

M

A

****p value show difference between base/impact study. p1 value show difference between 4 areas of vaccination in base/impact studies

23