LIVESTOCK PROTECTIVE FENCING TO PROTECT DAIRY CATTLE AGAINST VECTORS IN SERBIA - PROJECT PROCESSES AND METHODOLOGY

: Lumpy skin disease (LSD) is among a number of vector-borne diseases (VBDs) threatening the Balkans and therefore, preventing feeding of insects on cattle would reduce the spread of VBDs. In order to test the efficiency of Livestock Protective Fences (LPF) in the protection of dairy cattle from insect bites, a case-control study was conducted, in the districts of Nišava, Pirot and Pčinja, Southern Serbia. It consisted in comparing the number of biting flies collected within time, between 10 farms protected with LPF and 10 non protected ones. The insects were collected using two types of traps; the monoconical Vavoua trap set outside in between forested areas or rivers and the actual farm, and the BG-sentinel trap baited with CO 2 , placed in proximity of the cattle but outside the stable. Vectors were collected every 15 days for 48 hours from May to October 2018 and kept in vials containing 70% of ethanol. Catches per trap were separately


Introduction
Lumpy skin disease (LSD) is among a number of vector-borne diseases (VBDs) threatening the Balkans. This notifiable disease has dramatic effects on rural livelihoods and the effect at national level is also devastating due to strict trade restrictions Molla et al., 2017). The situation worsens with the arrival of summer's higher temperatures, favouring insect multiplication and, hence, disease dissemination. Therefore, preventing feeding of insects on livestock would also reduce the spread of VBDs (Bauer et al., 2006;Bauer et al., 2011). Unfortunately, vector control strategies used so far are either costly, time consuming or environmentally unacceptable. The large-scale and indiscriminate use of insecticides, mostly pyrethroids, constitutes the mainstay of vector control efforts. Pyrethroids are all upsetting the function of the sodium channels. Resistance against one pyethroid will lead to resistance against the whole class. This has led to widespread pyrethroid insecticides resistance in target vectors insects like Aedes sp., Culex sp. or Culicoides sp. (Laveissiere and Grebaut,1990;Caputo,2018;Pichler et al., 2018;Bengoa et al., 2017) Stable flies (Stomoxys spp.) have found to be less or no longer susceptible, (Reissert-Oppermann et al., 2019). However, there is a highly effective technology, the use of insecticide-incorporated knitted textile screens (known as livestock protective fences -LPF), which has been very successfully used in sub-Saharan African countries for the control of several VBDs (Heilmann et al., 2017;Maia et al., 2012;2010;Bauer et al., 2011). The fences are deployed in the vicinity of resting places near to cattle or alongside milking parlors, preventing insects from alighting on the animals.
The technology, never used in the Balkans, could be very useful in preventing VBDs. Another externality is stress reduction associated with insect bites of dairy cattle, an improvement of animal welfare and an increase in milk production (Maia et al., 2010). Moreover, significant reductions in mastitis cases have been recorded in the past, as insect vectors commonly transmit bacteria from animal to animal.
The current study aims to scientifically prove the efficiency of LPFs in the protection of dairy cattle from insect bites, thus reducing the transmission of VBD such as LSD, and reducing the stress of the animals due to insect bites.
If proven successful, the pilot study could be expanded within Serbia and to other affected and at-risk countries in the region .

Study location
Twenty farms were selected for a case-control study over one vector season, from mid-May to October 2018. Half of the farms were protected by LPF (case group), while the other half was considered as a control group, enabling to assess the trends of insect numbers and species. Half (50%) of the selected farms was located in the Nišava District with almost 100% of the case farms, and in Pirot and Pčinja districts, where control farms were mainly located ( Figure 1). All farms were dairy production ones and were mostly maintained on a zero-grazing scheme, with cattle number not exceeding 40 in total.
The farms were characterized through a questionnaire covering the production system, dung management, insect control treatments, etc.

T0 data collection and monitoring of the LPF impact on vector densities
Once a farm was selected, and before the deployment of the LPF, two types of traps were used to estimate the presence of target insect species: Monoconical Vavoua (Laveissière et al., 1990) (Figure 2) and BG-sentinel trap ( Figure 3). The Monoconical traps were set outside in between forested areas or rivers and the actual farm, while the BG-sentinel trap, baited with CO 2 (to enhance their efficacy) were placed in proximity of the cattle, but outside the stable, connected to a power source and attached to a CO 2 bottle. All trap locations were geo-referenced and visibly labelled. Throughout the intervention the positions of the traps were not supposed to be modified unless in a situation of emergency (change of pen location, bushfire, threat of theft). Continuous trapping would also allow assessing the LPF impact before and during intervention. Likewise, data on milk parameters, notably, the CFU (colony-forming units; i.e. the number of viable bacterial/fungal cells/ml) and the number of somatic cells were collected from the dairy association, for comparison. These data related to milk production should allow to assess the effects of LPF on animal welfare and common dairy associated diseases such as mastitis.

Impregnation of LPF
Lambda-Cyhalothrin in a suspension concentrate (S.C.) in a concentration of 0.6% supplied from Changzhou Biochemical Co. Ltd, China, in 10 litre canisters was used for the impregnation. For each impregnation, the netting material (100 m *0.8m; 1-2 mm diameter holes) was soaked in 2.7 litres of solution of active ingredient and water, to thoroughly moisten the fabric with as little as possible relic solution remaining. After impregnation, the netting was first dried and then attached to horizontal pieces of wood to facilitate deployment ( Figure 4).
In case farms, LPF were deployed covering all windows and openings in the stable, except for those needed for the operation of the farm, e.g. to get manure/feed in/out of the stable, and doors for animals and personnel to enter/exit/move around ( Figure 5). In addition, LPF are also deployed around dung pits or ponds when not covered.

Vector collection and identification
Vectors were collected every 15 days for 48 hours from May to October 2018 by the farmers and then sent to the Scientific Veterinary Institute in Belgrade in vials containing 70% of ethanol. Any collected insect was duly labeled and identified based on morphological characteristics, using keys given (Wall and Shearer, 1997;Cedric, 2005;Capinera 2008). Catches per trap were separately stored and, for each trap, insects were divided/classified according to species and sex and then counted.

Data analysis
An ANOVA was first applied to the data, and for each treatment, (control or case), the period (month) was used to explain the variation of insect numbers random effects. Data were then submitted to a multiple comparison of means (Tukey contrast) using statistical package software available online (R Development Core Team, Vienna, Austria). Because (case and control) are very different in terms of geographical distribution, and so cannot be compared by pairs, this comparison was done within each type of treatment (case and control). The insects were first pooled by genus (Mosquitoes, Stomoxys, Tabanids, etc.) before being submitted to analysis.
For the data on milk parameters, analysis was made separately, on data collected within protected farms, before and after the LPF deployment, and on data without protection. As for cell numbers, CFU data were analyzed with a negative binomial generalized linear mixed model. The treatment was used as explanatory variable, and the farm ID and months were considered as random effects

Insects collected
A total of 3,007 insects were collected during the 5-month monitoring period (Table 1), with up to 46% of Musca spp and 20% of Ceratopogonidae. Mosquitoes constitute up to 12% of the total insects caught, with a predominance of the genus Culex (54%) Aedes and Phlebotominae were only by BG traps (inside and closer to cattle) and none by the monoconical traps deployed outside and far from the cattle. Likewise, BG traps caught up 4-8 folds more Culex than monoconical traps.

Impact of LPF on vector densities
As indicated previously, this has been assessed in comparing the evolution of the densities within each type of treatment. Regardless of the natural variations, vector populations are expected to be decreasing within the time in farms where LPF was deployed. Numbers of different species of mosquitoes were pooled for this purpose.
For each of the species caught by both type of traps, no significant difference was observed between the two periods (before and after LPF deployment) in terms of insects' number.

Impact of LPF on milk quality parameters
In a first step, comparison was made within the 10 case farms before and after, showing a significant decrease of the number of CFU (a mean number of 319 to 122); p=0.028, figure 5.

Figure 5. Mean number of CFU in protected farms, before and after the LPD deployment
For the somatic cells, however, the decrease is not significant; 241 vs. 200, p=0.519 (figure 6).
In a second step, the comparison was made within the 10 control farms, no significant evolution was observed for both CFU (275 vs. 181, p=0.795) and cells (253 vs. 296, p=0.442).

Discussion
Target insect densities were recorded and compared on 20 farms in southern Serbia. Ten farms served as controls and 10 farms benefited from a protection with insecticide-impregnated netting material that had been strategically deployed, closing windows and entrances. This approach was aiming at the control of putative vectors for LSD and other VBDs. Vector control may serve as a prevention measure against VBDs.
Insect densities were monitored by using a total of forty traps -i.e. two trap types per farm. Mono-conical ("Vavoua") traps were deployed outside the farm buildings but still within the premises of the respective farm. Mono-conical farms are particularly suited for catching horse flies and muscid flies -including stomoxyine biting flies, commonly known as stable flies.
BG sentinel traps are known for their efficacy of catching mosquito populations. Also, they have been used on a large scale for assessing other hematophagous vectors such as ceratopogonidae (biting midges) and phlebotominae (sandflies). BG traps were deployed in proximity of the cattle inside the farms. All traps were controlled at fortnightly intervals and all catches were transferred to Belgrade (Scientific Veterinary Institute) for identification and counting.
A cotton wool netting material was impregnated with lambda-cyhalothrin, a pyrethroid formulation. Previous work using this approach had shown great potential of controlling target insect populations in various African countries (Bauer et al., 2011) and in Germany. The ready-to-use fence was then attached to a wooden frame, reducing the space for entering target insect species. The trial lasted for five months during 2018.
As in previous studies, no impact on vector densities was found. Usually, it would take at least two years for any detectable reduction of target vector species (Maia et al., 2010). However, when controlling tsetse flies in a forest area of South-Eastern Ghana significant differences between protected and unprotected pens were detected (Bauer et al., 2011).
The year 2018 was particular in much of Europe with temperatures reaching or exceeding 40 ○ C and a long, dry season. The climate may have had a negative impact on the survival rate of many insects. All the same, the initial figures of insect catches should have been high. However, at no time the numbers of collected insects corresponded to what has been recorded elsewhere. Indeed, during the Bluetongue outbreak in Germany, more than 1,000 Culicoides/trap/night were caught. It is puzzling and not well understood from our point of view why the total insect numbers were so low. For instance, it would be beyond any expectation from previous studies if a total of 603 biting midges were caught during five months with 40 traps. It is also puzzling to observe that the catches of midges with monoconical traps exceeded those ones recorded with BG sentinel traps. At the same time the catches of stable flies with mono-conical traps were widely below current expectations and cannot be explained at our level. It was also noted with surprise and cannot be explained why BG sentinel traps caught the vast majority of stable flies.
As already stated, any detectable impact on target vector populations would appear premature. However, it would be important and it is recommended to monitor and evaluate the efficacy of the netting material: immediately after impregnation and then at regular intervals up to the termination of this study.
During a study on dairy farms in Kenya, significant reductions of mastitis cases in milking cows due to their protection with LPF were shown. Similar assessment should be performed during an eventual follow-up study.
In view of the particular animal husbandry management system -all cattle were mostly kept inside the pens on a zero-grazing scheme, the putative vector populations can be narrowed down to stable flies, mosquito species, biting midges and sandflies. Arguably, the role of house flies should be considered as irrelevant in the transmission of LSD. Should there be a follow-up study, it might be worthwhile to record (with cameras) and compare defensive cattle movements on protected and control farms. Benefits would be more convincing if it were shown that the cattle well-being is greatly enhanced by a partial protection of their pens. Horse flies are exophile and exophagic. Their preference of feeding on horses and cattle while on pasture has been frequently observed. In reaction to this, it is observed for instance, that horses were trying to protect themselves by retreating under an open but roofed hut, whenever peak densities of horse flies were occurring. Stable flies, on the other hand readily feed on their hosts inside as well as outside the pens. Camera recordings of defensive movements would therefore go a long way in producing the required evidence for the benefits of protection.

Conclusion
The objective of this study was to explore the possibility of using LPF to protect cattle, in order to minimize risks of contracting VBD, notably LSD, and, also, to improve their wellbeing and milk quality. Although it was not possible to capture the direct impact of LPF on vector densities, the CFU values have improved. Some corrections/adaption in the methodology used may lead to better impact.